Ifn-alpha/beta-independent mechanism of antiviral protection through a novel ligand-receptor pair: ifn- ligands engage a novel receptor ifn-rn (crf2-12) and il-10r2 (crf2-4) for signaling andinduction of biological activities

ABSTRACT

A novel IFN-α/β independent ligand receptor system which upon engagement leads, among other things, to the establishment of an anti-viral state is disclosed. Further disclosed are three closely positioned genes on human chromosome 19 that encode distinct but highly homologous proteins, designated IFN-λ1, IFN-λ2, IFN-λ3, based, inter alia, in their ability to induce antiviral protection. Expression of these proteins is induced upon viral infection. A receptor complex utilized by all three IFN-λ proteins for signaling is also disclosed. The receptor complex is generally composed of two subunits, a novel receptor designated IFN-λR1 or CRF2-12, and a second subunit, IL-10R2 or CRF2-4, which is also a shared receptor component for the IL-10 and IL-22 receptor complexes. The gene encoding IFN-λR1 is generally widely expressed, including many different cell types and tissues. Expression of these proteins is induced by immune events, including, for example, upon viral infection. Apoptotosis may also be induced under effective conditions.

This application claims priority to provisional application entitled“New Human Class II Cytokine Receptor” Ser. No. 60/355,196 filed on Feb.8, 2002 and provisional application Ser. No. 60/418,474, entitledIFN-α/β-independent mechanism of antiviral protection through a novelligand-receptor pair: IFN-α ligands engage a novel receptor IFN-λR1(CRF2-12) and IL-10R2 (CRF24) for signaling and induction of biologicalactivities filed Oct. 15, 2002, both of which are incorporated herein byreference in their entirety, including the figures.

STATEMENT OF GOVERNMENT GRANT

This invention was partially funded by a grant from the United StatesGovernment AIS1139 from the National Institute of Allergy and InfectiousDiseases and therefore the United States Government may have rights inthis invention.

FIELD OF THE INVENTION

The present invention relates to three novel human genes encodingrelated polypeptides that are members of the interferon family and anovel receptor and receptor complex responsive to these novelinterferons. More specifically, isolated nucleic acid molecules areprovided encoding human polypeptides named IFN-λ1, IFN-λ2 and IFN-λ3 andIFN-λR1. Polypeptides are also provided, as are vectors, host cells andrecombinant methods for producing the same. Also provided are diagnosticmethods for detecting disorders and therapeutic methods for treatingdisorders. The invention further relates to screening methods foridentifying agonists and antagonists of IFN-λ1, IFN-λ2 and IFN-λ3 andIFN-λR1.

BACKGROUND OF THE INVENTION

Cytokines are small soluble regulatory molecules of the mammalian immunesystem. They control the growth, function and differentiation of a widevariety of cells and are therefore at the heart of all processes inwhich the body defends itself from infection of any nature. Banyer, etal., (2000) Rev Immunogenet 2, 359-373. Recently six new ligands withlimited homology to IL-10 have been identified. (Reviewed in Kontenko,et al., (2002)). Only a very limited amount is known about the functionof these IL-10 homologous ligands. Jiang et al, 1995, reported cloningmelanoma differentiation associated gene 7 (mda-7), a IL-10 homolog as aprotein whose expression is elevated in terminally-differentiated humanmelanoma cells. Soo et al, 1999 reported expression of the rat mda-7paralog was linked to wound healing; Zhang et al, 2000 designated theprotein c49a and linked it to ras transformation. The protein has alsobeen designated mob-5. The expression of rat mda-7 (c49a) was localizedprimarily to fibroblast-like cells at the wound edge and base. Soo, etal, 1999 further reported that during wound healing the level of c49amRNA was transiently elevated 9 to 12-fold above unwounded controls. Inaddition, expression of rat mda-7 (mob-5) was demonstrated to be inducedby expression of oncogenic ras. Moreover, mob-5 and its putativereceptor are oncogenic ras specific targets; mob-5 binds to the cellsurface of ras-transformed cells but not of parental untransformedcells. Zhang et al, 2000.

Saeki, et al., Oncogene 21(29):4558-66 (2002), reported that in additionto the overexpression of the melanoma differentiation associated gene-7(mda-7) in vitro resulting in suppression of lung cancer cellproliferation, overexpression of the mda-7 gene in human non-small celllung carcinoma cells in vivo has an effect on tumor growth. Inparticular, Saeki, et al., reported that adenovirus-mediatedoverexpression of MDA-7 in p53-wild-type A549 and p53-null H1299subcutaneous tumors resulted in significant tumor growth inhibitionthrough induction of apoptosis, and that decreased CD31/PECAM expressionand upregulation of APO2/TRAIL were observed in tumors expressing MDA-7.According to the authors, the data demonstrates that Ad-mda7 functionsas a multi-modality anti-cancer agent, possessing both pro-apoptotic andanti-angiogenic properties and thus may potentially serve as atherapeutic agent against human lung cancer.

Knappe, et al., J Virol 74(8):3881-7 (2000) reported cloning anotherIL-10 homolog, designated ak155, as a protein expressed by herpesvirussaimiri-transformed T lymphocytes.

Gallagher, et al., Genes Immun1 (7):442-50 (2000) reported theidentification and cloning of a gene and corresponding cDNAs encoding ahomologue of IL-10, which they designated IL-19. According to Gallagher,et al., IL-19 expression was induced in monocytes by Salmonellalipopolysaccharide (LPS) treatment, but IL-19 did not bind or signalthrough the canonical IL-10 receptor complex, suggesting existence of anIL-19 specific receptor complex, the identity of which remained to bediscovered.

Blumberg, et al., Cell 104(1):9-19 (2001), also reported cloning aprotein with homology to IL-10, which they designated Zcyto10 (GenBankaccession number AF224266) or IL-20. Several lines of evidencedemonstrate that IL-20 may play a functional role in epidermaldevelopment and psoriasis.

Dumoutier, et al., J Immunol 164(4):1814-9 (2000) reported cloningIL-TIF (IL-10 related T cell-derived inducible factor), an IL-10homolog, expressed by IL-9 treated murine T cells. Dumoutier, et al.,Proc Natl Acad Sci USA 97(18):10144-9 (2000) and Xie, et al., J BiolChem 275(40):31335-9 (2000) reported cloning IL-TIF's human orthologue(human IL-22). According to these reports, murine IL-22 expression canbe induced by IL-9 in thymic lymphomas, T cells and mast cells in vitroand by LPS in various organs in vivo, and IL-TIF injection inducedproduction of acute phase reactants in mouse liver, suggestinginvolvement of IL-TIF in the inflammatory response.

With the sequence of the human genome being completed, cytokines andreceptors have been discovered “in silico.” Currently there are 11members of the class II cytokine receptor family with assignedfunctions. Kotenko, S. V. (2002) Cytokine Growth Factor Rev. 13,223-240; Kotenko, et al., (2000) Oncogene 19, 2557-2565; Dumoutier, etal., (2002) Eur Cytokine Netw. 13, 5-15; Fickenscher, et al., (2002)Trends Immunol 23, 89-96. These receptors are primarily utilized forsignaling by members of two cytokine families, IFN and IL-10. The IFNfamily is further divided to type I and type II IFNs. The type II groupis represented by a single member, IFN-γ, whereas the type I group iscomprised of 13 IFN-α species (Data were obtained from the website ofthe National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/) with the use of various software toolsfor analyzing genome and EST databases available at the site.), a singlespecies of IFN-β, IFN-ω and IFN-κ (LaFleur, et al., (2001) J Biol Chem276, 39765-39771), and a mouse cytokine designated limitin (Oritani, etal., (2000) Nat. Med. 6, 659-666) for which a human homolog has not yetbeen identified (and may not exist).

All known type I IFN genes are clustered on human chromosome 9. There isat least one additional member of this family positioned in the samecluster.

The family of IL-10-related cytokines consists of six members ofcellular origin, IL-10, IL-19, IL-20, IL-22, IL-24 and IL-26, as well asseveral viral cytokines (for review see Kotenko, S. V. (2002) CytokineGrowth Factor Rev. 13, 223-240; Dumoutier, et al., (2002) Eur CytokineNetw. 13, 5-15; Fickenscher, et al., (2002) Trends Immunol 23, 89-96).

All of these cytokines possess important biological activities. Severalmembers of the IL-10 family are involved in a complex regulation ofinflammatory responses. Whereas anti-inflammatory activity of IL-10 (onmacrophages) is well characterized (Moore, et al., (2001) Annu. RevImmunol 19, 683-765), the functions of the other IL-10-related cytokinesare not well defined. IL-22 upregulates expression of several acutephase proteins in liver and hepatoma cells, and induces expression ofpancreatitis-associated protein (PAP1) in pancreatic acinar cells(Dumoutier, et al., (2000) Proc Natl Acad Sci USA 97, 10144-10149;Aggarwal, et al., (2001) J Interferon Cytokine Res 21, 1047-1053).

IL-20 seems to be involved in regulation of normal skin developmentbecause IL-20-transgenic mice display skin abnormalities characterizedby altered epidermal differentiation with hypoproliferation ofkeratinocytes (Blumberg, et al., (2001) Cell 104, 9-19).

The activities of IL-19 and IL-24 are not well characterized. Howeverthe fact that they share receptors with IL-20 and IL-22 indicates thatthey may share at least a subset of biological activities as well. Noinformation about the factional activities or the receptor for IL-26 iscurrently available.

Type I and type II IFNs are important immunomodulators. Type II orimmune IFN (IFN-γ) is a Th1-type cytokine which regulates both innateand adaptive immunity. IFN-γ stimulates cell-mediated immune responseswhich are critical for mediating protection against infection byintracellular parasites infections (many viruses) and is a part ofantiviral defense.

Type I IFNs are well known for their ability to induce antiviralprotection in wide variety of cells. They also activate host adaptiveand innate immune forces to eliminate viral infections.

Antiviral protection is a highly complex process which involves severallevels of defense starting with efforts of an infected cell to preventthe replication of a virus and warn other cells about viral presence,and finishing with the engagement of an entire army of immune protectiveforces to combat virus propagation in a body. It seems that the maintask of a first set of cells undergoing primary infection is to releasesignals to neighboring cells and immune system in general to alert themabout viral presence and to force them to build antiviral protectionbefore a virus infects them. Thus, the next wave of infected cells arebetter prepared to fight and upon sensing the presence of a virusthrough various means activate intracellular mechanisms of antiviralprotection or even commit suicide (apoptosis) to prevent viralreplication (reviewed in Levy, et al., (2001) Cytokine Growth Factor Rev12, 143-156 and Samuel, C. E. (2001) Clin Microbiol Rev 14, 778-809).These alert signals are also produced by a subset of dendritic cells,which are able to sense the presence of a virus through mechanisms notnecessarily involving viral entry into the cells. It is always a racebetween viral replication and development of cellular and immune defenseforces that determines severity, duration and outcome of a viralinfection.

Type I IFNs have been known for their ability to induce antiviralprotection on both cellular and immune levels. Virus-induced robustproduction and secretion of IFNs leads to the induction of expression ofmany proteins with antiviral actions. The most well studied proteins inthis respect are double-stranded RNA-activated protein kinase (PKR),2′,5′-oligodenylate synthetase (OAS) and Mx proteins. TheseIFN-inducible proteins prevent viral replication through variousmechanisms. dsRNA-activated PKR phosphorylates translation-initiationfactor eIF-2α blocking protein synthesis. OAS activates Rnase L whichcleaves mRNA and rRNA, thus inhibiting viral replication on bothtranscriptional and translational levels. Clearly, such drastic measuresaffect cellular viability; indeed, both enzymes have been implicated inapoptosis.

Mx proteins are GTPases and some of them possess antiviral activity. Themechanism of their action is not completely understood. Mx proteins arefound associated with viral ribonucleoprotein complexes and caninterfere with their transcriptional functioning and/or trafficking.

Several other IFN-inducible proteins are likely to participate incellular antiviral protection through mechanisms which remains to bedetermined. IFNs also modulate function of several type of immune cells(NK, CD-8+ and DC cells) in the direction favorable for clearing viralinfection (reviewed in Biron, C. A. (2001) Immunity 14, 661-664 and LeBon, A. & Tough, D. F. (2002) Curr. Opin. Immunol 14, 432-436).

There is a certain pattern of a ligand-receptor complex compositionwithin the IFN and IL-10 families (Kotenko, 2002). A specific receptorcomplex for a particular ligand from these families is composed of twodifferent receptor chains with distinct functions. The receptor chainswithin a given receptor complex can be divided into two types denoted R1and R2 based on their functions in signaling. Although both R1 and R2receptors are associated with Jak tyrosine kinases, only R1 typesubunits have a long intracellular domain, are phosphorylated on Tyrresidues after receptor engagement, therefore drive recruitment ofvarious signaling molecules and, thus, determine the specificity ofcytokine signaling. R2 type subunits possess a short intracellulardomain and support signaling by brining tyrosine kinase to a receptorcomplex but do not determine the specificity of signaling. The length ofthe CRF2-12 intracellular domain indicated that this receptor representsthe R1 type subunit.

There are five known receptor chains which can combine to form two-chainreceptors for one or more of the six known ligands of the IL-10 family,but these five known receptors do not by themselves allow the fullpotential repertoire of IL-10 homologue signaling to be realized. Thatis, there are too many ligands and not enough receptors. Furthermore andmost importantly, the wide and well-characterized in vivo and in vitroactivities of cytokines and their receptors acting in concertdemonstrated a clear need for and clinical potential of novel cytokines,cytokine receptors, cytokine mimics, blockers, agonists and antagonists.The present invention addresses these needs directly.

Type I IFNs are thought to exert their biological activities throughbinding to the specific cell surface receptor complex composed of twochains IFN-αR1 and IFN-αR2c (reviewed in Domanski, et al., (1996)Cytokine Growth Factor Rev 7, 143-151). IFN-αR2c is a signal-competentsplice variant encoded by the IFNAR2 gene. Studies with IFN-α/β receptorknockout mice in which either subunit of the IFN-α receptor complex havebeen disrupted demonstrate an essential role for IFN-α signaling in theinduction of antiviral resistance (Steinhoff, et al., (1995) J Virol 69,2153-2158; Muller, et al., (1994) Science 264, 1918-1921; Hwang, et al.,(1995) Proc Natl Acad Sci USA 92, 11284-11288). However, the loss ofantiviral protection to different viruses is variable in IFN-α receptorknockout mice. For instance, infection with rotavirus proceeds similarlyin mice with disrupted or intact IFN-α receptor system (Angel, et al.,(1999) J Interferon Cytokine Res 19, 655-659).

IFNs appear to share a common receptor mechanism, the type I IFN-Rcomposed of IFNAR1 and IFNAR2 subunits. IFNAR2 has membrane bound formsthat can be short or long and soluble forms. IFN induced receptordimerization of the IFNAR1 and IFNAR2c chains initiates a signalingcascade that involves tyrosine phosphorylation of the Tyk2 and Jak1tyrosine kinases and subsequent phosphorylation of the STAT1 and STAT2proteins (Stark et al., Ann. Rev. Biochem. 67:227-64 (1998); Science296:1632-1657 (2002)). Association of the phosphorylated STATs with thep48 DNA binding subunit, forms the ISGF3 multisubunit complex thattranslocates to the nucleus and binds to interferon-stimulated responseelements (ISRE) found upstream of the interferon inducible genes. Whilethe type I IFNs bind the same receptor there appears to be subsequentsignaling differences. In contrast to the type I IFNs there is only onemember of the type II IFN, namely IFN gamma, which is encoded by asingle gene (containing three introns) located on chromosome 12. Theprotein is produced predominantly by T lymphocytes and NK cells, is 166amino acids in length and shows no homology to type I interferons.

A range of biological activities are associated with IFNs includingantiviral, anti-microbial, tumor anti-proliferative, anti-proliferative,enhancement of NK cell activity, induction of MHC class I expression,and immunoregulatory activities. IFN alpha is marketed by ScheringPlough (Intron; IFN alpha 2B) and Hoffman La Roche (Roferon; IFN alpha2A). Therapeutic uses include the treatment of Hairy Cell leukemia,Chronic myelogenous leukemia, low grade non-Hodgkin lymphoma, cutaneousT cell lymphoma carcinoid tumors, renal cell carcinoma, squamousepithelial tumors of the head and neck, multiple myeloma, and malignantmelanoma. With regards to viral disease, Interferon alpha has been foundto aid the treatment of chronic active hepatitis, caused by eitherHepatitis B or C viruses. IFN Beta has been demonstrated to haveclinical benefit in the treatment of multiple sclerosis. Clinical trialswith Interferon gamma have shown potential in the treatment of cutaneousand also visceral leishmanias.

Both recombinant interferons and interferons isolated from naturalsources have been approved in the United States for treatment ofauto-immune diseases, condyloma acuminatum, chronic hepatitis C, bladdercarcinoma, cervical carcinoma, laryngeal papillomatosis, fungoidesmycosis, chronic hepatitis B, Kaposi's sarcoma in patients infected withhuman immunodeficiency virus, malignant melanoma, hairy cell leukemiaand multiple sclerosis.

Members of the type I interferon family have also been shown toinfluence neural cell activity and growth (see, for example, Dafny etal., Brain Res. 734:269 (1996); Pliopsys and Massimini,Neuroimmunomodulation 2:31 (1995)). In addition, intraventricularinjection of neural growth factors has been shown to influence learningin animal models (see, for example, Fischer et al., Nature 329:65(1987)).

IFNs have been used clinically for anti-viral therapy, for example, inthe treatment of AIDS (HIV infection) (Lane, Semin. Oncol. 18:46-52(1991)), viral hepatitis including chronic hepatitis B, hepatitis C(Woo, M. H. and Brunakis, T. G., Ann. Parmacother, 31:330-337 (1997);Gibas, A. L., Gastroenterologist, 1:129-142 (1993)), hepatitis D,papilloma viruses (Levine, L. A. et al., Urology 47:553-557 (1996)),herpes (Ho, M., Ann. Rev. Med. 38:51-59 (1987)), viral encephalitis(Wintergerst et al., Infection, 20:207-212 (1992)), respiratorysyncytial virus, panencephalitis, and other therapies, for example,mycosis fungoides and in the prophylaxis of rhinitis and respiratoryinfections (Ho, M., Annu. Rev. Med. 38:51-59 (1987)).

IFNs have been suggested for anti-parasite therapy, for example,IFN-gamma for treating Cryptosporidium paryum infection (Rehg, J. E., J.Infect. Des. 174:229-232 (1996)).

Anti-bacterial: IFNs have been used clinically for anti-bacterialtherapy. For example, IFN-gamma has been used in the treatment ofmultidrug-resistant pulmonary tuberculosis (Condos, R. et al., Lancet349:1513-1515 (1997)).

Interferon therapy has been used in the treatment of numerous cancers(e.g., hairy cell leukemia (Hoffmann et al., Cancer Treat. Rev. 12(Suppl. B): 33-37 (1985)), acute myeloid leukemia (Stone, R. M. et al.Am. J. Clin. Oncol. 16:159-163 (1993)), osteosarcoma (Strander, H. etal., Acta Oncol. 34:877-880 (1995)), basal cell carcinoma (Dogan, B. etal., Cancer Lett. 91:215-219 (1995)), glioma (Fetell, M. R. et al.,Cancer 65: 78-83 (1990)), renal cell carcinoma (Aso, Y. et al. Prog.Clin. Biol. Res. 303:653-659 (1989)), multiple myeloma (Peest, D. etal., Br. J. Haematol. 94:425-432 (1996)), melanoma (Ikic, D. et al.,Int. J. Dermatol. 34:872-874 (1995)), myelogenous leukemia, colorectalcancer, cutaneous T cell lymphoma, myelodysplastic syndrome, glioma,head and neck cancer, breast cancer, gastric cancer, anti-cancer vaccinetherapy, and Hodgkin's disease (Rybak, M. E. et al., J. Biol. ResponseMod. 9:14 (1990)). Synergistic treatment of advanced cancer with acombination of alpha interferon and temozolomide has also been reported(Patent publication WO 9712630 to Dugan, M. H.).

IFNs have been used clinically for immunotherapy or more particularly,for example, to prevent graft vs. host rejection, or to curtail theprogression of autoimmune diseases, such as arthritis, multiplesclerosis, or diabetes. IFN-beta is approved of sale in the UnitedStates for the treatment (i.e., as an immunosuppressant) of multiplesclerosis. Recently it has been reported that patients with multiplesclerosis have diminished production of type I interferons andinterleukin-2 (Wandinger, K. P. et al., J. Neurol. Sci. 149: 87-93(1997)). In addition, immunotherapy with recombinant IFN-alpha (incombination with recombinant human IL-2) has been used successfully inlymphoma patients following autologous bone marrow or blood stem celltransplantation, that may intensify remission following translation(Nagler, A. et al., Blood 89: 3951-3959 (June 1997)).

The administration of IFN-gamma has been used in the treatment ofallergies in mammals (See, International Patent Publication WO 8701288to Parkin, J. M. and Pinching, A. J.). It has also recently beendemonstrated that there is a reduced production of IL-12 andIL-12-dependent IFN-gamma release in patients with allergic asthma (vander Pouw Kraan, T. C. et al., J. Immunol. 158:5560-5565 (1997)). Thus,IFN may be useful in the treatment of allergy by inhibiting the humoralresponse.

Interferons may be used as an adjuvant or coadjuvant to enhance orsimulate the immune response in cases of prophylactic or therapeuticvaccination (Heath, A. W. and Playfair, J. H. L., Vaccine 10:427-434(1992)), such as in anti-cancer vaccine therapy.

Interferons have been used to treat corneal haze.

SUMMARY OF THE INVENTION

The present invention provides an isolated nucleic acid moleculecomprising a polynucleotide having a nucleotide sequence, for example,including: a nucleotide sequence encoding the CRF2-12 polypeptide havingthe complete amino acid sequence of SEQ ID NO: 2; a nucleotide sequenceencoding the CRF2-12 polypeptide having the complete amino acid sequenceof SEQ ID NO: 2 excepting the N-terminal methionine (i.e., residues2-520 of SEQ ID NO: 2); a nucleotide sequence encoding the matureCRF2-12 polypeptide shown as residues P19-520 of SEQ ID NO: 2; anucleotide sequence encoding the CRF2-12 polypeptide extracellulardomain; a nucleotide sequence encoding the extracellular domain of theCRF2-12 protein in plasmid pEF-CRF2-12/γR1; a nucleotide sequenceencoding the extracellular domain of the CRF2-12 protein in plasmidpEF3-FLγR2/αR2c; a nucleotide sequence encoding the intracellular domainof the CRF2-12 protein in plasmid pEF-FLCRF2-12; and a nucleotidesequence complementary to any of these nucleotide sequences.

The present invention also provides an isolated nucleic acid moleculecomprising a polynucleotide having a nucleotide sequence, for example,including: a nucleotide sequence encoding a chimeric receptor comprisingthe extracellular domain of CRF2-12 and the intracellular domain ofanother membrane bound receptor; a nucleotide sequence encoding achimeric receptor comprising the extracellular domain of CRF2-12 and theintracellular domain of another membrane bound tyrosine kinase receptor;a nucleotide sequence encoding a chimeric receptor comprising theextracellular domain of CRF2-12 and the intracellular domain of acytokine receptor; a nucleotide sequence encoding a chimeric receptorcomprising the extracellular domain of CRF2-12 and the intracellulardomain of an IFN R1 type receptor; a nucleotide sequence encoding achimeric receptor comprising the extracellular domain of CRF2-12 and theintracellular domain of IFNγR1; a nucleotide sequence encoding thechimeric protein in plasmid pEF-CRF2-12/γR1; and a nucleotide sequencecomplementary to any of these nucleotide sequences.

The present invention also provides an isolated nucleic acid moleculecomprising a polynucleotide having a nucleotide sequence, for example,including: a nucleotide sequence encoding a chimeric receptor comprisingthe intracellular domain of CRF2-12 and the extracellular domain ofanother membrane bound receptor; a nucleotide sequence encoding achimeric receptor comprising the intracellular domain of CRF2-12 and theextracellular domain of another membrane bound tyrosine kinase receptor;a nucleotide sequence encoding a chimeric receptor comprising theintracellular domain of CRF2-12 and the extracellular domain of acytokine receptor, a nucleotide sequence encoding a chimeric receptorcomprising the intracellular domain of CRF2-12 and the extracellulardomain of an IFN R1 type receptor; a nucleotide sequence encoding achimeric receptor comprising the intracellular domain of CRF2-12 and theextracellular domain of IL-10R1; a nucleotide sequence encoding thechimeric protein in plasmid pEF-IL-10R1/IFN-λR1; and a nucleotidesequence complementary to any of these nucleotide sequences.

The present invention also provides an isolated nucleic acid moleculecomprising a polynucleotide having a nucleotide sequence, for example,including: a tandem vector having a nucleotide sequence encoding tworeceptors, wherein the first receptor is an R1 type receptor and thesecond receptor is an R2 type receptor, and wherein the expression ofeach receptor is controlled by separate promoters and polyadenylationsignals; a tandem vector having a nucleotide sequence encoding tworeceptors, wherein the first receptor comprises the extracellular domainof CRF2-12 and the second receptor is an R2 type receptor, and whereinthe expression of each receptor is controlled by separate promoters andpolyadenylation signals; a tandem vector having a nucleotide sequenceencoding two receptors, wherein the first receptor comprises theintracellular domain of CRF2-12 and the second receptor is an R2 typereceptor, and wherein the expression of each receptor is controlled byseparate promoters and polyadenylation signals; a tandem vector having anucleotide sequence encoding two receptors, wherein the first receptorcomprises CRF2-12 and the second receptor is an R2 type receptor, andwherein the expression of each receptor is controlled by separatepromoters and polyadenylation signals; any of the above tandem vectors,wherein the R2 type receptor comprises CRF24; the tandem vectorpEF-CRF2-12/γR1+CRF2-4; and a nucleotide sequence complementary to anyof these nucleotide sequences.

The present invention also provides isolated nucleic acid moleculescomprising a polynucleotide encoding at least a portion of one of theIFNλ polypeptides having the complete amino acid sequence shown of SEQID NOS: 8, 10 and 12.

The nucleotide sequence determined by sequencing the IFN-λ1 clone, whichis shown in FIG. 16 (SEQ ID NO: 7), contains an open reading frameencoding a full length polypeptide of 200 amino acid residues, includingan initiation codon encoding an N-terminal methionine.

The nucleotide sequence determined by sequencing the IFN-λ2 clone, whichis shown in FIG. 17 (SEQ ID NO: 9), contains an open reading frameencoding a full length polypeptide of 196 amino acid residues, includingan initiation codon encoding an N-terminal methionine.

The nucleotide sequence determined by sequencing the IFN-λ3 clone, whichis shown in FIG. 18 (SEQ ID NO: 11), contains an open reading frameencoding a full length polypeptide of 196 amino acid residues, includingan initiation codon encoding an N-terminal methionine.

Nucleic acid molecules of the invention include those encoding thecomplete amino acid sequence excepting the N-terminal methionine shownin SEQ ID NOS: 8, 10 and 12, which molecules also can encode additionalamino acids fused to the N-terminus of the amino acid sequences ofIFN-λ1, IFN-λ2 and IFN-λ3.

Nucleic acid molecules of the invention include those encoding thecomplete amino acid sequence excepting the N-terminal methionine shownin SEQ ID NOS: 8, 10 and 12, which molecules also can encode additionalamino acids fused to the N-terminus of the amino acid sequences ofIFN-λ1, IFN-λ2 and IFN-λ3.

The encoded polypeptides have a predicted leader sequence of 22 aminoacids, shown as the boxed consensus in FIG. 19A.

Thus, one aspect of the invention provides an isolated nucleic acidmolecule comprising a polynucleotide comprising a nucleotide sequence,for example, including: a nucleotide sequence encoding the IFN-λ1polypeptide having the complete amino acid sequence of SEQ ID NO: 8; anucleotide sequence encoding the IFN-λ1 polypeptide having the completeamino acid sequence of SEQ ID NO: 8 excepting the N-terminal methionine(i.e., residues 2-200 of SEQ ID NO: 8); a nucleotide sequence encodingthe mature IFN-λ1 polypeptide shown as residues 23-200 of SEQ ID NO: 8;a nucleotide sequence of the genomic fragment encoding the completeIFN-λ1 gene contained in plasmid pEF-FL-IFN-λ1 gene; a nucleotidesequence encoding the complete polypeptide encoded by the cDNA containedin plasmid pEF-FL-IFN-λ1; a nucleotide sequence encoding the maturepolypeptide encoded by the human cDNA contained in plasmidpEF-FL-IFN-λ1; a nucleotide sequence encoding the mature polypeptideencoded by the human cDNA contained in plasmid pEF2-FL-IFN-λ1; and anucleotide sequence complementary to any of these nucleotide sequences.

Another aspect of the invention provides an isolated nucleic acidmolecule comprising a polynucleotide comprising a nucleotide sequence,for example, including: a nucleotide sequence encoding the IFN-λ2.1polypeptide having the complete amino acid sequence of SEQ ID NO: 10; anucleotide sequence encoding the IFN-λ2.1 polypeptide having thecomplete amino acid sequence of SEQ ID NO: 10 excepting the N-terminalmethionine (i.e., residues 2-196 of SEQ ID NO: 10); a nucleotidesequence encoding the mature IFN-λ2.1 polypeptide shown as residues23-196 of SEQ ID NO: 10; a nucleotide sequence encoding IFN-λ2.2polypeptide (residues M12-196 of SEQ ID NO: 10); a nucleotide sequenceencoding IFN-λ2.2 polypeptide residues A13-196 of SEQ ID NO: 10; anucleotide sequence encoding the mature IFN-λ2.2 polypeptide shown asresidues P31-196 of SEQ ID NO: 10; a nucleotide sequence encodingIFN-λ2.3 polypeptide (residues M92-196 of SEQ ID NO: 10); a nucleotidesequence encoding IFN-λ2.3 polypeptide residues A93-196 of SEQ ID NO:10; a nucleotide sequence encoding the mature IFN-λ2.3 polypeptide shownas residues P113-196 of SEQ ID NO: 10; the nucleotide sequence of thegenomic fragment encoding the complete IFN-λ2 gene contained in plasmidpEF-FL-IFN-λ2gene; a nucleotide sequence encoding a polypeptide encodedby the cDNA contained in plasmid pEF-FL-IFN-λ2; a nucleotide sequenceencoding the mature polypeptide encoded by the cDNA contained in clonepEF-FL-IFN-λ2; and a nucleotide sequence complementary to any of thesenucleotide sequences.

Another aspect of the invention provides an isolated nucleic acidmolecule comprising a polynucleotide comprising a nucleotide sequence,for example, including: a nucleotide sequence encoding the IFN-λ3.1polypeptide having the complete amino acid sequence of SEQ ID NO: 12; anucleotide sequence encoding the IFN-λ3.1 polypeptide having thecomplete amino acid sequence of SEQ ID NO: 12 excepting the N-terminalmethionine (i.e., residues 2-196 of SEQ ID NO: 12); a nucleotidesequence encoding the mature IFN-λ3.1 polypeptide shown as residuesP23-196 of SEQ ID NO: 12; a nucleotide sequence encoding IFN-λ3.2polypeptide (residues M6-196 of SEQ ID NO: 12); a nucleotide sequenceencoding IFN-λ3.3 polypeptide (residues M12-196 of SEQ ID NO: 12); anucleotide sequence encoding IFN-λ3.2 polypeptide residues P7-196 of SEQID NO: 12; a nucleotide sequence encoding IFN-λ3.3 polypeptide residuesA13-196 of SEQ ID NO: 12; a nucleotide sequence encoding the matureIFN-λ3.2 polypeptide shown as residues P31-196 of SEQ ID NO: 12; thenucleotide sequence of the genomic fragment encoding the complete IFN-λ3gene contained in plasmid pEF-FL-IFN-λ3gene; a nucleotide sequenceencoding a polypeptide encoded by the cDNA contained in plasmidpEF-FL-IFN-λ3; a nucleotide sequence encoding a mature polypeptideencoded by the cDNA contained in plasmid pEF-FL-IFN-λ3; and a nucleotidesequence complementary to any of these nucleotide sequences.

Further embodiments of the invention include isolated nucleic acidmolecules that comprise a polynucleotide having a nucleotide sequence atleast 80%, 85%, or 90% identical, more preferably at least 91%, 92%,93%, and 94% and most preferably at least 95%, 96%, 97%, 98% or to anyof the nucleotide sequences, above, or a polynucleotide which hybridizesunder stringent hybridization conditions to a polynucleotide of thepresent invention. This polynucleotide of the present invention, whichhybridizes under stringent conditions defined herein does not hybridizeto a polynucleotide having a nucleotide sequence consisting of only Aresidues or of only T residues. An additional nucleic acid embodiment ofthe invention relates to an isolated nucleic acid molecule comprising apolynucleotide that encodes the amino acid sequence of anepitope-bearing portion of a polypeptide having an amino acid sequencedescribed above.

A further aspect of the invention is a DNA sequence that represents thecomplete regulatory region of a gene of the present invention. DNAconstructs containing the regulatory region are also provided. Further,host cells comprising such constructs, which cells are in vitro or invivo, are also encompassed by the present invention.

The present invention also relates to recombinant vectors, which includethe isolated nucleic acid molecules of the present invention, to hostcells containing the recombinant vectors, as well as to methods ofmaking such vectors and host cells and for using them for production ofpolypeptides or peptides of the present invention by recombinanttechniques.

The invention further provides isolated polypeptides comprising an aminoacid sequence, for example, including: the amino acid sequence of thefull-length IFN-λ1 polypeptide having the complete amino acid sequenceof SEQ ID NO: 8; the amino acid sequence of the full-length IFN-λ1polypeptide having the complete amino acid sequence of SEQ ID NO: 8excepting the N-terminal methionine (i.e., residues 2-200 of SEQ ID NO:8); the amino acid sequence the mature IFN-λL polypeptide shown asresidues 23-200 of SEQ ID NO: 8; an IFN-λ1 polypeptide encoded by thegenomic fragment encoding the IFN-λ1 gene contained in plasmidpEF-FL-IFN-λ1 gene; an IFN-λ1 polypeptide encoded by the cDNA containedin plasmid pEF-FL-IFN-λ1; a mature IFN-λ1 polypeptide encoded by thecDNA contained in plasmid pEF-FL-IFN-λ1. The polypeptides of the presentinvention also include polypeptides having an amino acid sequence atleast 80% identical, more preferably at least 90% identical, and stillmore preferably 95%, 96%, 97%, 98% or 99% identical to those describedabove, as well as polypeptides having an amino acid sequence with atleast 90% similarity, and more preferably at least 95% similarity, tothose above.

The invention further provides isolated polypeptides comprising an aminoacid sequence, for example, including: the IFN-λ2.1 polypeptide havingthe complete amino acid sequence of SEQ ID NO: 10; the IFN-λ2.1polypeptide having the complete amino acid sequence of SEQ ID NO: 10excepting the N-terminal methionine (i.e., residues 2-196 of SEQ ID NO:10); the mature IFN-λ2.1 polypeptide shown as residues 23-196 of SEQ IDNO: 10; the IFN-λ2.2 polypeptide shown as residues M12-196 of SEQ ID NO:10; the IFN-λ2.2 polypeptide shown as residues A13-196 of SEQ ID NO: 10;the mature IFN-λ2.2 polypeptide shown as residues P31-196 of SEQ ID NO:10; the IFN-λ2.3 polypeptide shown as residues M92-196 of SEQ ID NO: 10;the IFN-λ2.3 polypeptide shown as residues A93-196 of SEQ ID NO: 10; themature IFN-λ2.3 polypeptide shown as residues P113-196 of SEQ ID NO: 10;a IFN-λ2 polypeptide coded by plasmid pEF-FL-IFN-λ2gene; a polypeptideencoded by the cDNA contained in plasmid pEF-FL-IFN-λ2; a maturepolypeptide encoded by the cDNA contained in clone pEF-FL-IFN-λ2. Thepolypeptides of the present invention also include polypeptides havingan amino acid sequence at least 80% identical, more preferably at least90% identical, and still more preferably 95%, 96%, 97%, 98% or 99%identical to those described above, as well as polypeptides having anamino acid sequence with at least 90% similarity, and more preferably atleast 95% similarity, to those above.

The invention further provides isolated polypeptides comprising an aminoacid sequence, for example, including: a IFN-λ3.1 polypeptide having thecomplete amino acid sequence of SEQ ID NO: 12; a IFN-λ3.1 polypeptidehaving the complete amino acid sequence of SEQ ID NO: 12 excepting theN-terminal methionine (i.e., residues 2-196 of SEQ ID NO: 12); a matureIFN-λ3.1 polypeptide shown as residues P23-196 of SEQ ID NO: 12; aIFN-λ3.2 polypeptide shown as residues M6-196 of SEQ ID NO: 12; aIFN-λ3.3 polypeptide shown as residues M12-196 of SEQ ID NO: 12; aIFN-λ3.2 polypeptide shown as residues P7-196 of SEQ ID NO: 12; aIFN-λ3.3 polypeptide shown as residues A13-196 of SEQ ID NO: 12; amature IFN-λ3.2 polypeptide shown as residues P31-196 of SEQ ID NO: 12;a IFN-λ3 polypeptide coded by plasmid pEF-FL-IFN-λ3gene; a polypeptideencoded by the cDNA contained in plasmid pEF-FL-IFN-λ3; a maturepolypeptide encoded by the cDNA contained in clone pEF-FL-IFN-λ3. Thepolypeptides of the present invention also include polypeptides havingan amino acid sequence at least 80% identical, more preferably at least90% identical, and still more preferably 95%, 96%, 97%, 98% or 99%identical to those described above, as well as polypeptides having anamino acid sequence with at least 90% similarity, and more preferably atleast 95% similarity, to those above.

An additional embodiment of this aspect of the invention relates to apeptide or polypeptide which comprises the amino acid sequence of anepitope-bearing portion of a polypeptide of the present invention havingan amino acid sequence described above. Peptides or polypeptides havingthe amino acid sequence of an epitope-bearing portion of a polypeptideof the invention include portions of such polypeptides with at least sixor seven, preferably at least nine, and more preferably at least about30 amino acids to about 50 amino acids, although epitope-bearingpolypeptides of any length up to and including the entire amino acidsequence of a polypeptide of the invention described above also areincluded in the invention.

In another embodiment, the invention provides an isolated antibody thatbinds specifically to a polypeptide having an amino acid sequencedescribed above. The invention further provides methods for isolatingantibodies that bind specifically to a polypeptide having an amino acidsequence as described herein. Such antibodies are useful therapeuticallyas described below.

The invention also provides for pharmaceutical compositions comprising apolypeptide of the present invention. The invention also provides forpharmaceutical compositions comprising combinations of polypeptides ofthe present invention.

In one embodiment, the invention provides for a composition comprisingIFN-λ1.

In another embodiment, the invention provides for a compositioncomprising IFN-λ2.

In another embodiment, the invention provides for a compositioncomprising IFN-λ3.

In one embodiment, the invention provides for a composition comprising acombination of polypeptides of the present invention, wherein the ratioof IFN-λ1:IFN-λ2:IFN-λ3 is described by the formula m:n:o, wherein m maybe any number between 0 and 1000, n may be any number between 0 and 1000and o may be any number between 0 and 1000, and wherein at least one ofm, n or o is different from 0. The invention also provides for acomposition comprising a combination of polypeptides of the presentinvention, wherein the ratio of IFN-λ1:IFN-λ2:IFN-λ3 is described by theformula m:n:o, wherein m may be any number between 0 and 1000, n may beany number between 0 and 1000 and o may be any number between 0 and1000, and wherein at least two of m, n or o are different from 0. Theinvention further provides for a composition comprising a combination ofpolypeptides of the present invention, wherein the ratio ofIFN-λ1:IFN-λ2:IFN-λ3 is described by the formula m:n:o, wherein m may beany number between 0 and 1000, n may be any number between 0 and 1000and o may be any number between 0 and 1000, and wherein all three of m,n and o are different from 0. The compositions may be employed, forinstance, as a positive control an assays for IFN-λ activity, as apositive control for CRF2-12 receptor and receptor complex activity, oras a pharmaceutical composition to treat immune system-relateddisorders, and other disorders such as viral infection, parasiticinfection, bacterial infection, cancer, autoimmune disease, multiplesclerosis, lymphoma and allergy. Methods of treating individuals in needof polypeptides of the present invention are also provided.

The invention also provides for pharmaceutical compositions comprisingan antibody of the present invention. The invention also provides forpharmaceutical compositions comprising combinations of antibodies of thepresent invention. In one embodiment, the invention provides acomposition comprising a combination of anti-IFN-λ1 specific antibody,anti-IFN-λ2 specific antibody and anti-IFN-λ3 specific antibody, whereinthe ratio of anti-IFN-λ1 specific antibody: anti-IFN-λ2 specificantibody: anti-IFN-λ3 specific antibody is described by the formulax:y:z, wherein x may be any number between 0 and 1000, y may be anynumber between 0 and 1000 and z may be any number between 0 and 1000,and wherein at least one of x, y or z is different from 0. The inventionalso provides for pharmaceutical compositions comprising a combinationof anti-IFN-λ1 specific antibody, anti-IFN-λ2 specific antibody andanti-IFN-λ3 specific antibody, wherein the ratio of anti-IFN-λ1 specificantibody: anti-IFN-λ2 specific antibody: anti-IFN-λ3 specific antibodyis described by the formula x:y:z, wherein x may be any number between 0and 1000, y may be any number between 0 and 1000 and z may be any numberbetween 0 and 1000, and wherein at least two of x, y or z are differentfrom 0. The invention also provides for pharmaceutical compositioncomprising a combination of anti-IFN-λ1 specific antibody, anti-IFN-λ2specific antibody and anti-IFN-λ3 specific antibody, wherein the ratioof anti-IFN-λ1 specific antibody: anti-IFN-λ2 specific antibody:anti-IFN-λ3 specific antibody is described by the formula x:y:z, whereinx may be any number between 0 and 1000, y may be any number between 0and 1000 and z may be any number between 0 and 1000, and wherein allthree of x, y or z are different from 0. The compositions may beemployed, for instance, to treat immune system-related disorders, andother disorders such as viral infection, parasitic infection, bacterialinfection, cancer, autoimmune disease, multiple sclerosis, lymphoma andallergy. Methods of treating individuals in need of polypeptides of thepresent invention are also provided.

The invention further provides compositions comprising a polynucleotideor a polypeptide for administration to cells in vitro, to cells ex vivoand to cells in vivo, or to a multicellular organism. In certainparticularly preferred embodiments of this aspect of the invention, thecompositions comprise a polynucleotide for the expression of apolypeptide of the present invention in a host organism for use to treata disease. Particularly preferred in this regard is expression in ahuman patient for treatment of a dysfunction associated with aberrantendogenous activity of an interferon or a receptor thereof.

The present invention also provides a screening method for identifyingcompounds capable of enhancing or inhibiting a biological activity ofthe polypeptides of the present invention, which involves contacting areceptor which is activated by the polypeptides of the present inventionwith the candidate compound in the presence of a polypeptide of thepresent invention, assaying, for example, anti-viral activity or MHCexpression in the presence of the candidate compound and the polypeptideof the present invention, and comparing the activity to a standard levelof activity, the standard being assayed when contact is made between thereceptor and a polypeptide of the present invention in the absence ofthe candidate compound. In this assay, an increase in activity over thestandard indicates that the candidate compound is an agonist and adecrease in activity compared to the standard indicates that thecompound is an antagonist of the activity of the polypeptide of thepresent invention.

The present invention also provides a screening method for identifyingcompounds capable of enhancing or inhibiting a biological activity ofthe polypeptides of the present invention, which involves contacting areceptor which is activated by an IFN-λ with the candidate compound inthe presence and in the absence of the IFN-λ, assaying for receptoractivity in the presence of the candidate compound and the IFN-λ,assaying for receptor activity in the presence of the candidate compoundand the absence of the IFN-λ, and comparing the activity to a standardlevel of activity, the standard being assayed when contact is madebetween the receptor and the IFN-λ in the absence of the candidatecompound.

Expression of the polypeptides of the present invention can be regulatedby double stranded RNA as well as other cytokines.

Polypeptides and antibodies directed to those polypeptides are useful toprovide immunological probes for differential identification of a numberof disorders, particularly of the immune system, significantly higher orlower levels of expression of genes of the present invention may bedetected in certain tissues (e.g., cancerous and wounded tissues), cellsor bodily fluids (e.g., serum, plasma, urine, synovial fluid or spinalfluid) taken from an individual having such a disorder, relative to astandard gene expression level, i.e., the expression level of the genein healthy tissue from an individual not having the disorder. Thus, theinvention provides a diagnostic method useful during diagnosis of such adisorder, which involves: (a) assaying the expression level of an mRNAor polypeptide of the present invention in cells or body fluid of anindividual; (b) comparing the expression level with a standardexpression level, whereby an increase or decrease in the assayedexpression level compared to the standard expression level is indicativeof disorder.

An additional aspect of the invention is related to a method fortreating an individual in need of an increased level of interferonactivity in the body comprising administering to such an individual acomposition comprising a therapeutically effective amount of an isolatedpolypeptide of the invention, combinations thereof, or an agonistthereof, or administration of DNA encoding one or more effectivepolypeptides of the present invention.

A still further aspect of the invention is related to a method fortreating an individual in need of a decreased level of interferonactivity in the body comprising, administering to such an individual acomposition comprising a therapeutically effective amount of anantagonist. Preferred antagonists for use in the present invention areIFN-λ-specific antibodies. Also preferred are antibodies specific forCRF2-12. Also preferred are antibodies specific for the extracellulardomain of CRF2-12. Particularly preferred are monoclonal antibodies.

A still further aspect of the invention is related to a method fortreating viral, parasitic, cancerous, allergic and other medicalconditions wherein the compositions of the present invention areeffective.

A still further aspect of the invention is related to a method formodulating the function of immune cells, including, for example isotypeswitching.

Another aspect of the present invention is the characterization ofgenetic disorders defined by polymorphisms in the genes or chromosomalloci of the present invention.

In another embodiment, the compositions of the present invention serveas an adjuvant for vaccination.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the nucleotide sequence (SEQ ID NO: 1) of human CRF2-12cDNA, with capital letters representing a predicted coding sequence.

FIG. 2 shows a deduced amino acid sequence (SEQ ID NO: 2) for humanCRF2-12.

FIG. 3 shows the CRF2-12 gene is located on human chromosome 1, between20.2 million bases and 20.4 million bases, adjacent to and telomeric ofthe gene for IL22R1 receptor chain, in the cytogenic region1p36.11-1p36.12.

FIG. 4 shows an alignment between the amino acid sequence of theextracellular portion of CRF2-12 and that of IL22R1.

FIG. 5 shows an alignment of the CRF2-12 polypeptide (SEQ ID NO: 2) ofthe present invention with several other members of the IL-10 receptorpolypeptide family. Shown are (labeled IL10Ra), (labeled IL10Rb),(labeled IL20Ra), (labeled IL20Rb), (labeled IL22Ra), (labeled IL22BP).Amino acids identical to the CRF2-12 polypeptide are boxed. By examiningthe regions of the boxed amino acids, the skilled artisan can readilyidentify conserved domains between the polypeptides. These conserveddomains are preferred embodiments of the present invention.

FIG. 6 shows an alignment of the CRF2-12 polypeptide (SEQ ID NO:2) ofthe present invention with several other members of the class-IIcytokine receptor polypeptide family. Shown is (labeled ar2), (labeledtfec), (labeled crf28), (labeled crf210), labeled crf29), (labeledil10r1), (labeled ar1n), (labeled il10r2), (labeled ar1c), (labeledgr2), (labeled hudirs1ec), (labeled gr1). Amino acids identical to theCRF2-12 polypeptide are boxed. By examining the regions of the boxedamino acids, the skilled artisan can readily identify conserved domainsbetween the polypeptides. These conserved domains are preferredembodiments of the present invention.

FIG. 7 shows the nucleotide sequence (SEQ ID NO: 1) and the deducedamino acid sequence (SEQ ID NO: 2) of human CRF2-12.

FIG. 8 shows the nucleotide sequence (SEQ ID NO: 1) and the deducedamino acid sequence (SEQ ID NO: 2) of human CRF2-12 using analternative, three-letter, notation sometimes used to describe aminoacid sequences.

FIG. 9 represents the single letter nucleotide sequence (SEQ ID NO: 3)which fully describes and defines the codon usages possible for encodingthe deduced amino acid sequence (SEQ ID NO: 2) of human CRF2-12.

FIG. 10 shows how the redundant code defines possible individual codonsencoding the deduced amino acid sequence (SEQ ID NO: 2) of humanCRF2-12.

FIG. 11 shows the 3′UTR portion of CRF2-12 (SEQ ID NO: 4).

FIG. 12 shows the nucleotide sequence (SEQ ID NO: 5) of mouse CRF2-12cDNA, with capital letters representing a predicted coding sequence.

FIG. 13 shows a deduced amino acid sequence (SEQ ID NO: 6) for mouseCRF2-12.

FIG. 14A shows the deduced amino acid sequence (SEQ ID NO:2) of humanCRF2-12, where the first boxed region, region a, constitutes the signalsequence, the first non-boxed region, region b, constitutes thepredicted intracellular domain, the second boxed region, region c,constitutes the predicted transmembrane domain, and the second non-boxedregion, region d, constitutes the predicted extracellular domain ofhuman CRF2-12. Potential glycosylation sites are underlined as well asthe C-terminal intracellular Tyr-based motif which is also homologous tothose within the IFN-αR2c intracellular domain and is likely toparticipate in Stat activation.

FIG. 14B shows a phylogenetic tree derived from an alignment of theextracellular domains of the class II cytokine receptors, which assignsCRF2-12 to a subgroup with several other receptors including CRF2-8,CRF2-9, CRF2-10, IL-10R1 and IFN-γR1. Alignment was generated by theprogram PILEUP of the Wisconsin Package Version 9.1, Genetics ComputerGroup (GCG), Madison, Wis. was used with the following parameters: thegap creation penalty 1, the gap extension penalty 1. The CLUSTAL Xprogram was used to create the phylogenetic tree.

FIG. 14C shows the organization of the CRF2-12 gene. The gene iscomposed of 7 exons and its structure correlates well with the commonconserved architecture of other genes encoding CRF members. The firstexon contains the 5′-UTR and the signal peptide. Exons 2, 3, 4 and 5 anda part of exon 6 encode the extracellular domain. Exon 6 also encodesthe transmembrane domain and the beginning of the intracellular domain.Exon 7 covers the rest of the intracellular domain and the 3′-UTR.

FIG. 14D a schematic diagram of human chromosome 1, shows that theCRF2-12 gene and the IL-22R1 gene are transcribed in the same directiontoward the telomere and are positioned approximately 10 kb apart.Chromosomal localization of the CRF2-12 gene and its close neighbor, theIL-22R1 gene as well as the ideogram of human chromosome 1 was generatedfrom the NCBI database. The genes are transcribed in the same directionas indicated by the arrow. Schematic exon/intron structure of theCRF2-12 gene is shown. Coding regions of exons are shaded and thesegments corresponding to 5′ and 3′ untranslated regions are left open.

FIGS. 15A and B, as shown in these figures, CRF2-12 appears to beconstitutively expressed in a variety of cell lines and tissues. FIG.15A is a northern blot, and shows that CRF2-12 mRNA is expressed atvariable levels in all the tumor cell lines examined, includinghematopoietic (HL-60, K-562, MOLT-4 and Raji) and non-hematopoietic(HeLa, SW480, A549, and G-361) cell lines.

Northern blotting was performed on two blots containing mRNA isolatedfrom: (A) human cancer cell lines (promyelocytic leukemia HL-60,epitheloid carcinoma HeLa S3, lymphoblastic leukemia MOLT-4, Burkitt'slymphoma Raji, colorectal adenocarcinoma, SW480, lung carcinoma A549 andmelanoma G361) and (B) normal human fetal tissues (heart, kidney, skin,small intestine) and adult lung. Arrows pointing to the CRF2-12 and theb-actin transcripts. Equal RNA loading was assessed by evaluating theexpression of the b-actin gene.

FIG. 15B is a northern blot, and shows that CRF2-12 was also expressedby various normal tissues, including heart, kidney, skin, smallintestine, and lung. CRF2-12 mRNA was also present in skeletal muscleand liver.

FIG. 16 shows the nucleotide sequence (SEQ ID NO: 7) and the deducedamino acid sequence (SEQ ID NO: 8) of IFN-λ1.

FIG. 17 shows the nucleotide sequence (SEQ ID NO: 9) and the deducedamino acid sequence (SEQ ID NO: 10) of IFN-λ2.

FIG. 18 shows the nucleotide sequence (SEQ ID NO: 11) and the deducedamino acid sequence (SEQ ID NO: 12) of IFN-λ3.

FIG. 19A shows a sequence alignment of polypeptides IFN-λ1 (SEQ ID NO:8), IFN-λ2 (SEQ ID NO: 10) and IFN-λ3 (SEQ ID NO: 12). Identicalresidues are boxed. The consensus sequence is shown on the bottom.Positions of corresponding introns are indicated by arrows. By examiningthe regions of the boxed amino acids, the skilled artisan can readilyidentify conserved domains between the polypeptides. These conserveddomains are preferred embodiments of the present invention. Amino acid(a.a.) residues are numbered starting from the Met residue (signalpeptide a.a. are included).

FIG. 19B shows a phylogenetic tree for IFN-λ proteins generated asdescribed in the FIG. 14 legend.

FIG. 19C is a schematic showing the gene structure and chromosomallocalization for IFN-λ genes as well as the direction of transcription(arrows). Coding regions of exons are shaded and the segmentscorresponding to 5′ and 3′ untranslated regions are left open.

FIG. 20A, from left to right, is a diagrammatic illustration oftransmembrane receptors encoded by cDNA expression vectors encoding i)intact CRF2-12 ii) a chimeric CRF2-12/γR1 receptor that has the CRF2-12extracellular domain fused to the transmembrane and intracellulardomains of the human IFN-γR1 chain, iii) CRF2-4, the second chain of thehuman IL-10 and IL-22 receptor complexes and iv) a FLAG tagged chimericIL-10R1/CRF2-12 receptor that has the IL-10R1 extracellular domain fusedto the transmembrane and intracellular domains of CRF2-12.

FIG. 20B is a diagrammatic illustration of IFN-λs and their derivatives.From top to bottom, i) represents IFN-λs; ii) represents IFN-λs taggedat the N-terminus with the FLAG epitope (FL-IFN-λs); iii) representsIFN-λs tagged at the C-terminus with the Arg-Arg-Ala-Ser-Val-Alasequence that contains the consensus amino acid motif recognizable bythe catalytic subunit of the cAMP-dependent protein kinase(FL-IFN-λs-P).

FIG. 20C is a western blot showing the expression of FL-I IFN-λs andFL-IFN-λ1-P in COS cells. Western blotting analysis with anti-FLAGantibody of conditioned media from COS-1 cells transfected with plasmidspEF-SPFL (lane 1, mock), pEF-FL-IFN-λ1 gene (lane 2, FL-IFN-λ1 gene),pEF-FL-IFN-λ2gene (lane 3, FL-IFN-λ2gene), pEF-FL-IFN-λ3gene (lane 4,FL-IFN-λ3gene), pEF-FL-IFN-λ1 (lane 5, FL-IFN-λ1), pEF-FL-IFN-λ2 (lane6, FL-IFN-λ2), pEF-FL-IFN-λ3 (lane 7, FL-IFN-λ3). FL-IFN-λ1-α waspurified from conditioned media by affinity chromatography and evaluatedby Western blotting with anti-FLAG antibody (lane 8). Lane 9 representsan autoradiograph of the SDS-PAGE gel containing radiolabeledFL-IFN-λ1-P. The molecular weight markers are shown on the left.

FIG. 21 MHC class I antigen expression, ligand binding and EMSAperformed on hamster cells.

A. Four cell lines used in these experiments are schematically shown(top panels): the parental Chinese hamster 16-9 cells and cellsexpressing the human CRF2-12/gR1 chimeric receptor or human IL-10R2alone or both receptors together. The cells were left untreated (openareas, thick lines) or treated with conditioned media (100 ml) from COScells transfected with plasmid pEF-FL-IFN-λ1 (FL-IFN-λ1; shaded areas,thin lines) or with E. coli produced IFN-λ1 (IFN-λ1E. coli, 100 ng; openareas, thin lines) and the ability of IFN-λ1 to upregulate MHC class Iantigen expression was demonstrated by flow cytometry (panels A, B, Cand D). The cells were also tested for their ability to bind FL-IFN-λs(panels E, F, G, H and I). The cells were incubated for three hours at20° C. with conditioned medium from COS-1 cells transfected with one ofthe following plasmids: the control vector pEF-SPFL (open areas, thicklines); the pEF-FL-IFN-λ1 (FL-IFN-λ1; open areas, thin lines; panels E,F, G and H) or the pEF-FL-IFN-λ2 (FL-IFN-λ2; shaded areas, thin lines;panel I). Specificity binding was demonstrated by ligand bindingcompetition. Cells expressing both chains were co-incubated with eitherFL-IFN-λ1 or FL-IFN-λ2 (100 ml of conditioned media from COS cellsexpressing FL-IFN-λ1/2) and with E. coli produced IFN-λ1 lacking theFLAG epitope (IFN-λ1E. coli, 1 mg/ml) (open areas, thin lines; panels Hand I). Ligand binding to the cell surface was determined by flowcytometry with anti-FLAG antibody (Sigma) as the primary antibody andfluorescein isothiocyanate-conjugated goat anti-mouse IgG (Santa Cruz)as the secondary antibody. The ordinate represents relative cellnumbers, and the abscissa relative fluorescence (logarithmic scale).

FIG. 21B. The hamster cells described in FIG. 21A row 1 were leftuntreated or treated with various stimuli: conditioned media (100 ml)form COS cells transfected with plasmids pEF-FL-IFN-λ1/2/3 gene(FL-IFN-λsgene), pEF-FL-IFN-λ1/2/3 (FL-IFN-λs), PEF-IFN-λ1 (IFN-λ1 COS).Numbers 1, 2 and 3 in treatment lanes reflect which IFN-λ protein(IFN-λ1, IFN-λ2 and IFN-λ3, respectively) was used for the treatment. E.coli produced IFN-λ1 was also evaluated (IFN-λ1E. coli). Anti-CRF24antibody (1 mg) was added to the cells 30 minutes before IFN-λtreatment.

FIG. 22 shows the results of experiments wherein ligand was crosslinkedto hamster cells transfected with receptor subunit combinations.Untreated 32P-labeled FL-IFN-λ1-P ([32P]FL-IFN-λ1-P) was loaded as acontrol (lane 1). ([32P]FL-IFN-λ1-P) was also incubated in DMEM mediumwith 10% FBS and crosslinking in solution was performed as described(32) (lane 2). The untransfected and transfected hamster cells describedin FIG. 22 row 1 were incubated with 32P-labeled FL-IFN-λ1-P with orwithout addition of a 100-fold excess of unlabeled IFN-λ1 (competitor),washed, harvested and crosslinked. The extracted crosslinked complexeswere analyzed on 7.5% SDS-PAGE. The molecular weight markers are shownon the left.

FIG. 23A shows western blots, wherein the ability of chimericFL-IL-10R1/CRF2-12 receptor to activate Stat in response to IL-10 andIFN-α in HT-29 cells was analyzed.

FIG. 23B shows an EMSA gel, wherein modified ISGF3 binding to ISRE inresponse to IL-10 and IFN-α in HT-29 cells expressing the chimericFL-IL-10R1/CRF2-12 receptor was analyzed.

FIG. 23C shows EMSA gels, wherein A549 cells response to IFN-λ1 andIFN-α was measured by their ability to modify binding to GAS and ISREDNA probes as indicated. Positions of Stat DNA-binding complexes inEMSAs are indicated by arrows.

FIG. 23D are flow cytometry graphs showing the level of MHC class Iantigen expression in HeLa, HT29 and A549 in response to treatment withconditioned medium from COS cells transfected with plasmid pEF-FL-IFN-λ1(FL-IFN-λ1; shaded areas, thin lines) or treatment with Hu-IFN-a (100un/ml; open areas, thin lines) as compared to control cells leftuntreated (open areas, thick lines). The ordinate represents relativecell numbers, and the abscissa relative fluorescence (logarithmicscale).

FIGS. 24A and B one of several biological activities of IFN-λS. FIG. 24Ashows cell survival and antiviral protection in response to IFN-λ1. Anequal amount of cells were plated in all wells and treated with two-foldserial dilutions of ligands, as indicated (rows A and C). Twenty-fourhours later the virus was added in all wells except for the first sevenwells in row F. Row C shows that HT29 cells exhibit a dose dependentsurvival response to vesicular stomatitis virus (VSV) in response toadministration of increasing IFN-λ1 concentrations. Row A shows the dosedependent response to IFN-α, a positive control.

FIG. 24B shows the change in IFN-λs mRNA expression in virus-infectedcells. HeLa cells were left untreated (lane 3) or infected with eitherDengue virus (DV, lanes 4-6) Sindbis virus (SV, lanes 7-9) or vesicularstomatitis virus (VSV, lanes 10⁻¹²). HT29 cells were left untreated(lane 13) or infected with VSV (lanes 14-16). HuH7 cells were leftuntreated (lane 17) or infected with DV (lanes 18-20) or SV (lanes21-23). Cells were collected at post-infection times shown in theFigure, RNA was isolated and RT-PCR was performed as described inMaterials and Methods. Water was used as a negative control for RT-PCR(lane 2). 1 kb ladder was run in lanes 1 and 24.

FIG. 25 is a schematic model of IFN-λs' interaction with the CRF2-12receptor system. In one aspect of the invention IFN-λs are monomers. Inanother embodiment of the present invention, IFN-λs are multimers of andbetween the three members of the IFN-λ family. In another embodiment ofthe present invention a multimer may contain other members of thecytokine family, including the IFN and IL-10 families of cytokines. Afunctional IFN-λ receptor complex may include two receptor chains, forexample, the unique CRF2-12 (IFN-λR1) chain and the CRF2-4 (or IL-10R2)chain. The IL-10R2 chain is a shared common chain for at least threereceptor complexes, the IL-10, IL-22 and IFN-λ receptor complexes. Whenexpressed alone, neither chain of the IFN-λ receptor complex is capableof IFN-λ binding. Expression of both chains is required for ligandbinding and for assembling of the functional receptor complex. Ligandbinding leads to the formation of the heterodimeric receptor complex andto the initiation of a signal transduction cascade. The CRF2-4 chain isassociated with Tyk2 (Kotenko, S. V., Izotova, L. S., Mirochnitchenko,O. V., Esterova, E., Dickensheets, H., Donnelly, R. P. & Pestka, S.(2001) J Biol Chem 276, 2725-2732 and Levy, D. E. & Garcia-Sastre, A.(2001) Cytokine Growth Factor Rev 12, 143-156) and the IFN-λ R1 chain islikely to interact with Jak1. Upon the ligand-induced heterodimerizationof IFN-λ receptor chains receptor-associated Jaks crossactivate eachother, phosphorylate the IFN-λR1 intracellular domain and, thus,initiate the cascade of signal transduction events. Stat1, Stat2, Stat3and Stat5 are activated by IFN-λ leading to activation of biologicalactivities, such as upregulation of MHC class I antigen expression andinduction of antiviral protection.

FIG. 26. Antiviral response. (A) Antiviral protection in response toIFN-λ1 was evaluated on either HT29, A549 or HaCaT cells infected withvesicular stomatitis virus (VSV) using cytopathic effect (CPE) reductionassay performed in 96-well microtiter plate. IFN-α a was used as apositive control. Antiviral activity is shown as the amount of IFN-α orIFN-λ1 for 50% protection of the cells from CPE. (B) Expression ofISRE-controlled genes encoding 2′,5′-oligoadenylate synthetase(2=,5=-OAS) and MxA protein in response to IFN-α and IFN-λ1 wasevaluated by RT-PCR. Cells were treated for 16 h with indicated ligands,RNA was isolated and subjected to RT-PCR. (C) The hamster cellsexpressing IFN-λR1/λR1+IL-10R2 were left untreated or treated withvarious stimuli: conditioned media from COS cells containing FL-IFN-□;IFN-λ2 (1000 u/ml); and conditioned media from untreated HT29 cells(HT29) or EMCV-infected HT-29 or A549 cells (HT29+EMCV and A549+EMCV).Where indicated STAT1 Ab was added to shift the mobility of theSTAT-DNA-binding complexes.

FIG. 27 shows further amino acid sequences for IFN-λ1, IFN-λ2 andIFN-λ3.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides isolated nucleic acid moleculescomprising a novel family of cytokines designated IFN-λ, their specificfunctional receptor complex, and characterization of their signaling andbiological activity.

The present invention further provides two receptor proteins, CRF2-12and IL-10R2 (CRF24), that comprise a functional receptor complex forthese novel ligands.

The present invention further provides a novel IFN-α/β-independentligand-receptor system which upon engagement leads to the establishmentof an antiviral state.

All references cited herein are incorporated herein by reference intheir entirety, including the figures and sequences.

Three closely positioned genes on human chromosome 19 encode distinctbut highly homologous proteins which we designated IFN-λ1, IFN-λ2 andIFN-λ3 based to their ability to induce antiviral protection. Expressionof these proteins is induced upon viral infection. A receptor complexwhich is utilized by all three IFN-λ proteins for signaling is a furtherembodiment of the present invention. This complex is composed of twosubunits, a novel receptor designated IFN-λR1 or CRF2-12, and a secondsubunit, IL-10R2 or CRF2-4, which is also a shared receptor componentfor the IL-10 and IL-22 receptor complexes. Binding of IFN-λs to thereceptor complex induces signaling through the Jak-Stat signaltransduction pathway, including activation of the ISGF3 transcriptioncomplex, the same complex that is activated by signaling through theIFN-α/β receptor complex. Stat recruitment is largely mediated by theIFN-αR2c intracellular domain (Kotenko, S. V., Izotova, L. S.,Mirochnitchenko, O. V., Lee, C. & Pestka, S. (1999) Proc Natl Acad SciUSA 96, 5007-5012 and Prejean, C. & Colamonici, O. R. (2000) Semin.Cancer Biol 10, 83-92).

It is interesting to note that there is a Tyr-based motif(Tyr517MetAlaArgStop) at the C-terminus of the IFN-λR1 intracellulardomain which is also present at the end of the IFN-αR2c intracellulardomain (Tyr512IleMetArgStop) which is competent in Stat activationduring IFN-α signaling ((39); Y. Ge, S. V. K. and S. Pestkaunpublished). This motif is also conserved in mouse IFN-λR1(TyrLeuValArgStop)₃. Thus, the IFN-λ and IFN-α/β signaling pathways atleast partially overlap. As a consequence, at least some of theirbiological activities are similar, including induction of antiviralprotection and up-regulation of MHC class I antigen expression.According with their antiviral activity, the expression of IFN-λs mRNAsis induced by viral infections in several cell lines and in dendriticcells in response to polyI:C treatment.

The signal transduction pathways and biological activities of IFN-λs andIFN-α overlap. In another embodiment of the present invention, an IFN-λor a combination of IFN-λs may be used as a substitute, or incombination with, IFN-λ. In another aspect of the present invention,IFN-λs can synergize with IFN-α in mediating resistance to viralinfections (or plays an independent primary role in establishment ofresistance to certain viruses), and other conditions for which IFN-α iseffective.

In another embodiment of the present invention, an IFN-λ or acombination of IFN-λs may be used to treat, ameliorate or preventconditions for which IFN-α is not effective, for example, viralinfections of the gastrointestinal tract.

A new receptor protein designated as “CRF2-12”, one possible nucleotidesequence which can encode the complete CRF2-12 protein is FIG. 1 and thecomplete polypeptide CRF2-12 has the amino acid sequence in FIG. 2.Automated analysis of the protein sequence using the “PIX” package(http://www.hgmp.mrc.ac.uk) revealed the presence of the followingfeatures in the CRF2-12 polypeptide: region a) a leader sequence/signalpeptide comprising residues 1-20 or thereabouts; region b) anextracellular region between residues 20-228 or thereabouts; region c) atransmembrane region between residues 228-244 or thereabouts; and regiond) an intracellular region/cytoplasmic tail region between residues244-520 or thereabouts. The CRF2-12 gene is expressed in spleen,placenta and other tissues and is located on human chromosome 1, between20.2 million bases and 20.4 million bases, adjacent to and telomeric ofthe gene for IL22R1 receptor chain, in the cytogenic region1p36.11-1p36.12, as shown in FIG. 3. The present invention providesmethods for detecting genetic abnormalities in patients, developingknock-out mice or transgenically altered mice, see FIGS. 12 and 13,treatment of a variety of disorders including but not limited toinflammatory disorders, malignant disorders and disorders associatedwith autoimmunity. In addition, the invention includes diagnostic kitscomprising several antibodies such as ELISA kits, or ‘in situhybridization’. Finally, the invention includes therapeutic agents thatcomprise complementary cDNA, RNA sequences or variants thereof.

The 208 amino acid polypeptide fragment of CRF2-12 region b sharessignificant identity and similarity with the corresponding extracellularportion of members of the class-II cytokine receptor family. Asillustrated in FIG. 4, marked similarity exists between theextracellular portion of CRF2-12 and that of IL22R1. The 208 aminopolypeptide fragment of CRF2-12 region b therefore represents theextracellular domain of a new member of the class-II cytokine receptorfamily. The similarity between CRF2-12 and other members of the IL-10receptor family is shown in FIG. 5, while its broader relationship withother members of the class-II cytokine receptor family is shown in FIG.6.

As used herein, unless otherwise specified, the term cytokine includesproteins which are variously termed “interferons”, “interleukins”,“colony-stimulating factors”, “tumor necrosis factors” and so on.Indeed, these terms are frequently used interchangeably with the termcytokine. Cytokines are known to elicit biological activities viabinding to specific membrane-bound receptors.

As used herein, unless otherwise specified, the term ExtracellularDomains includes those of the receptors composed of one or moreproteins, which have proteins outside the cell.

As used herein, unless otherwise specified, the term TransmembraneDomains includes those of the receptors composed of one or moreproteins, which extend through the cell membrane.

As used herein, unless otherwise specified, the term IntracellularDomains includes those of the receptors composed of one or moreproteins, which extend into the cytoplasm of the cell.

The present invention describes and defines isolated polypeptides ofgreater than or equal to 70% identity to the amino acid referencesequence revealed in FIG. 2, covering both the whole polypeptide andspecific regions “a”, “b”, “c” and “d” as defined below.

Region “a”, to be residues 1-19, 1-20, 1-21 and/or 1-22, and may betermed herein the signal sequence.

Region “b” to be residues 19-226, 19-227, 19-228, 19-230, 20-226,20-227, 20-228, 20-229, 20-230, 21-226, 21-227, 21-228, 21-229, 21-230,22-226, 22-227, 22-228, 22-229, 22-230, and may be termed herein theextracellular domain.

Region “c” to be residues 226-242, 226-243, 226-244, 226-245, 226-246,227-242, 227-243, 227-244, 227-245, 227-246, 228-242, 228-243, 228-244,228-245, 228-246, 229-242, 229-243, 229-244, 229-245, 229-246, 230-242,230-243, 230-244, 230-245, 230-246, and may be termed herein thetransmembrane domain.

Region “d” to be residues 242-520, 243-520, 244-520, 245-520, 246-520,and may be termed herein the intracellular domain.

The present invention also describes and defines isolated polypeptidescomprising at least 12 contiguous amino acids of the polypeptidesequence shown in FIG. 2, selected from groups “a”, “b”, “c” and/or “d”.

In the present invention, “isolated” refers to material removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring). For example, an isolated polynucleotide could be part of avector or a composition of matter, or could be contained within a cell,and still be “isolated” because that vector, composition of matter, orparticular cell is not the original environment of the polynucleotide.The term “isolated” does not refer to genomic or cDNA libraries, wholecell total or mRNA preparations, genomic DNA preparations (includingthose separated by electrophoresis and transferred onto blots), shearedwhole cell genomic DNA preparations or other compositions where the artdemonstrates no distinguishing features of the polynucleotide/sequencesof the present invention.

In the present invention, a “secreted” protein of the present inventionrefers to a protein capable of being directed to the ER, secretoryvesicles, or the extracellular space as a result of a signal sequence,as well as a proteins of the present invention released into theextracellular space without necessarily containing a signal sequence. Ifthe secreted proteins of the present invention is released into theextracellular space, the secreted proteins of the present invention canundergo extracellular processing to produce a “mature” proteins of thepresent invention. Release into the extracellular space can occur bymany mechanisms, including exocytosis and proteolytic cleavage.

A “membrane” associated polypeptide of the present invention may beutilized as a polypeptide integrated in a lipid membrane, such as amembrane-bound polypeptide, an intracellular polypeptide expressed inthe cell's secretory pathway, a polypeptide expressed in the plasmamembrane at the cell surface or as a polypeptide integratedsynthetically into membrane-like structures such as in liposomes ormicelles.

The polynucleotides of the present invention can be composed of anypolyribonucleotide or polydeoxyribonucleotide, which may be unmodifiedRNA or DNA or modified RNA or DNA. For example, polynucleotides can becomposed of single- and double-stranded DNA, DNA that is a mixture ofsingle- and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that may be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions. In addition, the polynucleotides can be composed oftriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thepolynucleotides of the present invention may also contain one or moremodified bases or DNA or RNA backbones modified for stability or forother reasons. “Modified” bases include, for example, tritylated basesand unusual bases such as inosine. A variety of modifications can bemade to DNA and RNA; thus, “polynucleotide” embraces chemically,enzymatically, or metabolically modified forms.

Polypeptides of the present invention can be composed of amino acidsjoined to each other by peptide bonds or modified peptide bonds, i.e.,peptide isoesteres, and may contain amino acids other than the 20gene-encoded amino acids. The polypeptides of the present invention maybe modified by either natural processes, such as post-translationalprocessing, or by chemical modification techniques which are well knownin the art. Such modifications are well described in basic texts and inmore detailed monographs, as well as in a voluminous researchliterature. Modifications can occur anywhere in the polypeptides of thepresent invention, including the peptide backbone, the amino acidside-chains and the amino or carboxyl termini. It will be appreciatedthat the same type of modification may be present in the same or varyingdegrees at several sites in the polypeptides of the present invention.Also, the polypeptides of the present invention may contain many typesof modifications. Polypeptides of the present invention may be branched,for example, as a result of ubiquitination, and they may be cyclic, withor without branching. Cyclic, branched, and branched cyclic polypeptidesof the present invention may result from posttranslation naturalprocesses or may be made by synthetic methods. Modifications includeacetylation, acylation, ADP-ribosylation, amidation, covalent attachmentof flavin, covalent attachment of a heme moiety, covalent attachment ofa nucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cysteine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, pegylation, proteolytic processing,phosphorylation, prenylation, racemization, selenoylation, sulfationtransfer-RNA mediated addition of amino acids to proteins such asarginylation, and ubiquitination. (See, for instance, PROTEINS—STRUCTUREAND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman andCompany, New York (1993); POSTTRANSLATIONAL COVALENT MODIFICATION OFPROTEINS, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12(1983); Seifter et al., Meth Enzymol 182:626-646 (1990); Rattan et al.,Ann NY Acad Sci 663:48-62 (1992).)

Polypeptides of the present invention “having biological activity”refers to polypeptides exhibiting activity similar, but not necessarilyidentical to, an activity of a polypeptides of the present invention(e.g., anti-viral activity, ability to bind an antibody which binds apolypeptide of the present invention, ability to bind to CRF2-12, or asdescribed in the example section herein), including mature forms, asmeasured in a particular biological assay, with or without dosedependency. In the case where dose dependency does exist, it need not beidentical to that of the polypeptides of the present invention, butrather substantially similar to the dose-dependence in the activity ascompared to the polypeptides of the present invention (i.e., thecandidate polypeptide will exhibit greater activity or not more thanabout 25-fold less and, preferably, not more than about tenfold lessactivity, and most preferably, not more than about three-fold lessactivity relative to the polypeptides of the present invention.)

“Regulatory regions” is intended to include promoters, enhancers and/orother expression control elements (e.g., polyadenylation signals), whichmay be in any effective relationship with the coding sequence. Suchregulatory sequences are described, for example, in Goeddel, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). Regulatory sequences include those that directconstitutive expression of a nucleotide sequence in many types of hostcell and those that direct expression of the nucleotide sequence only incertain host cells (e.g., tissue-specific regulatory sequences). It willbe appreciated by those skilled in the art that the design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression of protein desired, etc.

IFN-λs are expressed mainly in all cells, and particularly in dentriticcells natural cytokine producing cells. Expression of the proteins andpolypeptides of the present invention may be regulated in any effectivemanner. For example, by double stranded RNA, anti-sense technology, aswell as cytokines and effective small molecules.

By upregulating the level of CRF2-12 expression in virus-infected cellsor in tumor cells we can force these cells undergo apoptosis orantiproliferative effect in response to IFN-λS. Enhanced expression ofCRF2-12 can be, perhaps, induced by some agents, or simply achieved bytargeted delivery of CRF2-12 expression construct into virus- or otherpathogen-infected cells, or malignant cells. CRF2-12 expression can bereplaced by expression of a chimeric receptor, such as IL-10R1/CRF2-12described above. The extracellular domain of such chimeric receptor canbe substituted by the extracellular domain of various receptors. If suchchimeric receptors are expressed in target cells (malignant cells orpathogen-infected cells), these cells can be killed by the treatmentwith the appropriate ligand which binds to the extracellular domain ofthe chimeric receptor and induce signaling through the CRF2-12intracellular domain. Some cancers express autocrine growth factors tomaintain cancer growth, or express certain cytokines such as IL-10 tosuppress the immune system. Thus, by using the extracellular domain ofan appropriate receptor in combination with the intracellular domain ofCRF2-12, cancer cells expressing such chimeric receptor can be killed bytheir own growth factors or cytokines. The extracellular domain ofIL-10R1 seems to be most useful since many cancers, particularlyadvanced cancers, produce high level of IL-10.

Therapeutic Indications

The first embodiment is the use of IFN-λs to induce antiviral protectionto treat viral infections in both humans and other mammals. Suchprotection may be induced for the treatment of an ongoing infection orfor prophylaxis of anticipated infections, including but not limited tocommon recurring infections such as influenza, and circumstancesrequiring emergency prophylaxis, such as a bioweapon attack.

Another embodiment is the use of IFN-λs to induce apoptosis to treathyperproliferative disorders in both humans and other mammals. And inaddition, to withdraw interferon lambda in order to prevent apoptosis,for the treatment of hypoproliferative disorders in humans and othermammals. It is understood that the stated administration or withdrawalof IFN lambda also included other methods of manipulation of the IFNlambda/receptor signaling mechanism which have similar effects to directligand manipulation. Such means might include, but are not limited toantibody moieties or other binding structures which bind the receptor,mimicking or blocking ligand effects and or interference with downstreamsignaling events resulting from receptor engagement. It will be realizedthat hypo and hyper proliferative disorders will include all humanmalignancies and other overgrowths such as benign tumors and cysts inaddition to disease causing or promoting systems which include theover-growth or under-growth of one or more individual cell types. Suchsystems may include any and all disorders of the immune system,including autoimmune diseases, the digestive system, the haematopoieticsystem, the nervous and neuronal system, the cardiovascular system, therespiratory system, tooth eruption and other such bodily mechanisms,such as growth and development of fetuses and juveniles where thenatural, regular and programmed selective growth and death of cells maybecome disrupted.

A further embodiment is the control of the glycosylation of IFN-λ1 as atarget for drug development to induce either an antiviral effect orinduce cell death to treat either humans or other mammals. Othermechanisms for manipulating the affinity or selective action ofinterferon lambda, such as changing of one or more amino acids of theIFN-lambda peptide sequence, or insertion or removal of short peptideelements to/from the natural IFN-lambda sequence are also envisaged.

Another embodiment is the use of IFN-λs to upregulate MHC class Iantigen expression and, thus, stimulate immune response against viruses,other pathogens and tumors in both humans and other mammals.

A final embodiment is the use of novel sequences in gene therapy totreat either humans or other mammals, including but not limited to thesubstitution of interferon lambda sequences from other mammals to thehuman sequence in order to manipulate the ligand affinity for itsnatural receptor, or to induce therapeutic signaling or blocking of anunrelated receptor. Furthermore, the construction of chimeric molecules,comprised of IFN lambda sequences and peptide sequenced from human orother mammalian cytokines, growth factors etc for the purpose oftherapy, research and/or teaching is also envisaged.

The present invention also includes variations of CRF2-12. It will berecognized by someone trained in the art that mutations can exist in apeptide sequence, which do not affect the function of the protein.Often, these occur naturally but can be produced artificially bysubstituting one or more amino acid residues for the natural ones. Suchso-called “conservative” substitutions include but are not limited to;the substitution of one acidic amino acid for another (e.g. Asparticacid for glutamic acid), one non-polar amino acid for another (e.g.Glycine for alanine), one hydrophobic amino acid for another (e.g.Leucine for isoleucine), one basic amino acid for another (e.g. Lysinefor arginine), and so on. Examples of amino acids in similar groups arewidely known and accepted in the field. Such conservative changes in thepeptide sequence therefore fall within the scope of this invention. Itwill be further recognized by someone trained in the art that mutations,resulting or created as above, can exist in a peptide sequence whichalter the function of a protein and/or its ability to interact withother proteins. In particular, the mutation may alter the ability of thereceptor to bind a natural ligand and/or alter the consequences ofligand binding by preventing the receptor from transmitting a signal orby enhancing the signal. Such non-conservative changes fall within thescope of this invention and are included in it.

The present invention also describes and defines isolated nucleic acidmolecules, which encode the CRF2-12 polypeptides, its fragments andmutants. An illustrative nucleic acid sequence which can encode CRF2-12is given in FIG. 1. This nucleic acid sequence will encode all theCRF2-12 fragments and peptides described in regions “a”, “b”, “c” and“d”. Translated products of nucleic acid sequences are usually quoted ina single-letter code, as shown in FIG. 2, and FIG. 7 illustrates thetranslation of the nucleic acid sequence revealed in FIG. 1. Analternative, three-letter, notation is sometimes used to describe thetranslation of nucleic acid sequence and this is shown in FIG. 8, forCRF2-12.

As is widely recognized in the art, there is redundancy in thetranslation of nucleic acid sequences. Often, several triplet codons maybe translated to the identical amino acid. The present inventionpresupposes that natural variation at any of the triplet codons mayresult in a nucleic acid sequence which varies at one or more positionsfrom the illustrative sequence revealed in FIG. 1 will still result inan identical translation product to that revealed in FIG. 2, indeed thatsuch variations are not only possible but likely, especially betweenindividuals from different ethnic backgrounds. Such redundancy will alsoapply to the natural or man-made conservative and/or non-conservativeamino-acid changes referred to above. The IUPAC convention for writingnucleic acid sequences allows for the use of single letters which maydefine two or more nucleotides, in addition to the four uniquenucleotides (A, C, G, T). These are widely known in the art. FIG. 9,reveals the single-letter nucleic acid sequence, which fully describesand defines the possible extent of variation in a gene encoding CRF2-12,while FIG. 10 expands this to show how the redundant code definespossible individual codons.

As is widely recognized in the art, portions of genes which encodepeptides (exons) are usually separated from one another by intervening,non-coding nucleic acid sequences (introns). Such introns are integralparts of the gene. The coding sequence for CRF2-12 revealed in FIG. 1 isdistributed within at least seven exons and six intervening introns. Thepresent invention incorporates these exons and introns to form part ofthe gene describing CRF2-12. Also widely recognized is the fact thatfollowing the coding portion of a gene comes a non-coding portion, whichis a part of the last exon, the so-called “3′UTR”. The present inventiondescribes and defines the 3′UTR for the CRF2-12 gene and this isrevealed in FIG. 11.

As is widely recognized in the art, multi-exon genes, such as the genefor CRF2-12, may exist as splice variants, where one or more codingexons are missing, resulting in a shorter translation product, or added,resulting in a translation product which can be either longer orshorter. The present invention provides for the naturally-occurringpresence of such splice variants and incorporates them. In its primaryform, the gene product CRF2-12 is expected to be found embedded withinthe membranes of cells. However, it will be recognized by thoseknowledgeable in the art that splice variants can occur which excludethe transmembrane and intracellular portions of the protein, resultingin a protein which is freed into the extracellular milieu. Such variantswould result in a soluble CRF2-12 and so soluble CRF2-12 is included inthe scope of this invention.

As is widely recognized in the art, individuals may vary in one or morenucleotide residues within a given gene, such as the CRF2-12 gene. Suchnatural variations are often referred to as “allelic variants.” Thesevariants are useful for the prediction and monitoring of diseases,particularly diseases involving CRF2-12 or adjacent genes.

As is common practice in the art, cell-bound receptor structures similarin nature to CRF2-12, are often produced from an artificiallyconstructed polynucleotide, such that the product of said nucleotidecomprises the specific invention (in this case the extracellular portionof CRF2-12 or parts thereof) operationally and functionally joined to aprotein or protein fragment with the property of improving thepharmacokinetics of the specific invention. A molecule frequently usedin this way is the so-called Fc region of human immunoglobulin,resulting in a chimeric molecule (in this case CRF2-12 and Fc) encodedfrom a single polynucleotide. An example of this in clinical use todayis the hybrid TNF(p75) receptor/Fc molecule. The present inventionanticipates the use of the CRF2-12 gene for the construction of suchchimeric polynucleotides, which are thereby incorporated in the scope ofthe invention.

As is common knowledge in the art, cell-surface receptors of the CRFfamily, such as the interleukin-10 receptor, the interleukin-20 receptorand the interleukin-22 receptor exist functionally as two or moreindividual polypeptide chains. Furthermore, it will be appreciated thatsynthetic chimeric polynucleotides can be produced which encode theextracellular portions of one or more of the known members of the CRFfamily (including, but not limited to: interleukin-10 receptor alpha,interleukin-10 receptor beta, interleukin-20 receptor alpha,interleukin-20 receptor beta, interleukin-22 receptor alpha, interferongamma receptor components, interleukin alpha/beta receptor components)in association with CRF2-12. Such chimeric polypeptides might representthe natural specificity of CRF2-12 or a novel specificity which dependupon the presence of the CRF2-12 polypeptide. The present inventiontherefore incorporates such chimeric molecules and their resultantspecificities where, in addition to CRF2-12 said chimeric polypeptidecontains sequences from one or more other members of the CRF family andconstitute novel, soluble receptor entities. It is anticipated that suchnovel soluble receptor entities would find use in the treatment ofdiseases described previously.

The present invention, CRF2-12 and its various embodiments, constitute areceptor for IFN-λ. The receptor nature of CRF2-12 will allow otherligands to be identified by processes commonly used in the art. Forexample, one aspect of the present invention includes a method ofscreening for CRF2-12 ligands which includes the steps of binding apolypeptide including CRF2-12 to a solid, inert matrix and passing overthe resultant complexes a biological mixture which contains a potentialCRF2-12 ligand, whereby the ligand is retained and then harvesting theligand in a purified form using methods well-established in the art(such as ion-exchange) and identified. Since the identification ofCRF2-12 ligand is wholly dependent upon the present invention, CRF2-12ligands are therefore included in the scope of the present invention.

Polypeptides and Fragments

The invention further provides an isolated IFN-λ1 polypeptide having theamino acid sequence encoded by pEF-FL-IFN-λ1 gene, an isolated IFN-λ1polypeptide having the amino acid sequence encoded by pEF-FL-IFN-λ1, anisolated IFN-λ1 polypeptide having the amino acid sequence in SEQ ID NO:8, or a peptide or polypeptide comprising a portion of the abovepolypeptides.

The invention further provides an isolated IFN-λ2 polypeptide having theamino acid sequence encoded by pEF-FL-IFN-λ2gene, an isolated IFN-λ2polypeptide having the amino acid sequence encoded by pEF-FL-IFN-λ2, anisolated IFN-λ2 polypeptide having the amino acid sequence in SEQ ID NO:10, or a peptide or polypeptide comprising a portion of the abovepolypeptides.

The invention further provides an isolated IFN-λ3 polypeptide having theamino acid sequence encoded by pEF-FL-IFN-λ3gene, an isolated IFN-λ3polypeptide having the amino acid sequence encoded by pEF-FL-IFN-λ3, anisolated IFN-λ3 polypeptide having the amino acid sequence in SEQ ID NO:12, or a peptide or polypeptide comprising a portion of the abovepolypeptides.

The invention further provides an isolated polypeptide comprising anamino-acid sequence of the full-length IFN-λ1 polypeptide having thecomplete amino acid sequence of SEQ ID NO: 8. The invention furtherprovides an isolated polypeptide comprising the amino acid sequence ofthe full-length IFN-λ1 polypeptide having the complete amino acidsequence of SEQ ID NO: 8 excepting the N-terminal methionine (i.e.,residues 2-200 of SEQ ID NO: 8). The invention further provides anisolated polypeptide comprising the amino acid sequence of the matureIFN-λ1 polypeptide shown as residues 23-200 of SEQ ID NO: 8. Theinvention further provides an isolated polypeptide comprising an IFN-λ1polypeptide encoded by the genomic fragment encoding the IFN-λ1 genecontained in plasmid pEF-FL-IFN-λ1 gene. The invention further providesan isolated polypeptide comprising an IFN-λ1 polypeptide encoded by thecDNA contained in plasmid pEF-FL-IFN-λ1. The invention further providesan isolated polypeptide comprising a mature IFN-λ1 polypeptide encodedby the cDNA contained in plasmid pEF-FL-IFN-λ1. The polypeptides of thepresent invention also include polypeptides having an amino acidsequence at least 80% identical, more preferably at least 90% identical,and still more preferably 95%, 96%, 97%, 98% or 99% identical to thosedescribed above, as well as polypeptides having an amino acid sequencewith at least 90% similarity, and more preferably at least 95%similarity, to those above.

The invention further provides an isolated polypeptide comprising theIFN-λ2 polypeptide having the complete amino acid sequence of SEQ ID NO:10. The invention further provides an isolated polypeptide comprisingthe IFN-λ2 polypeptide having the complete amino acid sequence of SEQ IDNO: 10 excepting the N-terminal methionine (i.e., residues 2-196 of SEQID NO: 10). The invention further provides an isolated polypeptidecomprising the mature IFN-λ2 polypeptide shown as residues 23-196 of SEQID NO: 10. The invention further provides an isolated polypeptidecomprising the IFN-λ2 polypeptide shown as residues M12-196 of SEQ IDNO: 10.

The invention further provides an isolated polypeptide comprising theIFN-λ2 polypeptide shown as residues A13-196 of SEQ ID NO: 10. Theinvention further provides an isolated polypeptide comprising the matureIFN-λ2 polypeptide shown as residues P31-196 of SEQ ID NO: 10. Theinvention further provides an isolated polypeptide comprising the IFN-λ2polypeptide shown as residues M92-196 of SEQ ID NO: 10. The inventionfurther provides an isolated polypeptide comprising the IFN-λ2polypeptide shown as residues A93-196 of SEQ ID NO: 10. The inventionfurther provides an isolated polypeptide comprising the mature IFN-λ2polypeptide shown as residues P113-196 of SEQ ID NO: 10. The inventionfurther provides an isolated polypeptide comprising a IFN-λ2 polypeptidecoded by plasmid pEF-FL-IFN-λ2gene.

The invention further provides an isolated polypeptide comprising apolypeptide encoded by the cDNA contained in plasmid pEF-FL-IFN-λ2. Theinvention further provides an isolated polypeptide comprising a maturepolypeptide encoded by the cDNA contained in clone pEF-FL-IFN-λ2. Thepolypeptides of the present invention also include polypeptides havingan amino acid sequence at least 80% identical, more preferably at least90% identical, and still more preferably 95%, 96%, 97%, 98% or 99%identical to those described above, as well as polypeptides having anamino acid sequence with at least 90% similarity, and more preferably atleast 95% similarity, to those above.

The invention further provides an isolated polypeptide comprising aIFN-λ3.1 polypeptide having the complete amino acid sequence of SEQ IDNO: 12. The invention further provides an isolated polypeptidecomprising a IFN-λ3 polypeptide having the complete amino acid sequenceof SEQ ID NO: 12 excepting the N-terminal methionine (i.e., residues2-196 of SEQ ID NO: 12). The invention further provides an isolatedpolypeptide comprising a mature IFN-λ3 polypeptide shown as residuesP23-196 of SEQ ID NO: 12. The invention further provides an isolatedpolypeptide comprising a IFN-λ3 polypeptide shown as residues M6-196 ofSEQ ID NO: 12. The invention further provides an isolated polypeptidecomprising a IFN-λ3 polypeptide shown as residues M12-196 of SEQ ID NO:12. The invention further provides an isolated polypeptide comprising aIFN-λ3 polypeptide shown as residues P7-196 of SEQ ID NO: 12. Theinvention further provides an isolated polypeptide comprising a IFN-λ3polypeptide shown as residues A13-196 of SEQ ID NO: 12. The inventionfurther provides an isolated polypeptide comprising a mature IFN-λ3polypeptide shown as residues P31-196 of SEQ ID NO: 12. The inventionfurther provides an isolated polypeptide comprising a IFN-λ3 polypeptidecoded by plasmid pEF-FL-IFN-λ3gene.

The invention further provides an isolated polypeptide comprising apolypeptide encoded by the cDNA contained in plasmid pEF-FL-IFN-λ3. Theinvention further provides an isolated polypeptide comprising a maturepolypeptide encoded by the cDNA contained in clone pEF-FL-IFN-λ3. Thepolypeptides of the present invention also include polypeptides havingan amino acid sequence at least 80% identical, more preferably at least90% identical, and still more preferably 95%, 96%, 97%, 98% or 99%identical to those described above, as well as polypeptides having anamino acid sequence with at least 90% similarity, and more preferably atleast 95% similarity, to those above.

An additional embodiment of this aspect of the invention relates to apeptide or polypeptide which comprises the amino acid sequence of anepitope-bearing portion of a polypeptide of the present invention havingan amino acid sequence described above. Peptides or polypeptides havingthe amino acid sequence of an epitope-bearing portion of a polypeptideof the invention include portions of such polypeptides with at least sixor seven, preferably at least nine, and more preferably at least about30 amino acids to about 50 amino acids, although epitope-bearingpolypeptides of any length up to and including the entire amino acidsequence of a polypeptide of the invention described above also areincluded in the invention.

In another embodiment, the invention provides an isolated antibody thatbinds specifically to a polypeptide having an amino acid sequencedescribed above. The invention further provides methods for isolatingantibodies that bind specifically to a polypeptide having an amino acidsequence as described herein. Such antibodies are useful therapeuticallyas described below.

To improve or alter the characteristics of the polypeptides of thepresent invention, protein engineering may be employed. In addition,recombinant DNA technology known to those skilled in the art, including,for example, site directed mutagenesis, can be used to create novelmutant proteins or “muteins” including single or multiple amino acidsubstitutions, deletions, additions or fusion proteins.

Modified polypeptides of the present invention can show, e.g., enhancedor decreased (i.e., blocking) activity or increased stability. Inaddition, they may be purified in higher yields, show better solubilitythan the corresponding natural polypeptide, and be more effectivetherapeutically, at least under certain purification, storage andadministration conditions.

Unless otherwise specified, a “polypeptide fragment” refers to an aminoacid having a sequence which is a portion of that contained in SEQ IDNO: 2, SEQ ID NO:8, SEQ ID NO: 10 or SEQ ID NO: 12, or is encoded by thenucleic acid sequence in plasmids pEF-CRF2-12/γR1+CRF24,pEF-IL-10R1/IFN-λR1, pEF-CRF2-12/R1, pEF-FLCRF2-12, pEF-CRF2-12,pEF-CRF2-12/γR1, pEF-FL-IFN-λ1, pEF-FL-IFN-λ2, pEF-FL-IFN-λ3,pEF-FL-IFN-λ1 gene, pEF-FL-IFN-λ2gene or pEF-FL-IFN-λ3gene.

The protein or polypeptide fragments of the present invention may becomplete within itself, or it may be comprised within a largerpolypeptide of which the fragment forms a part or region, mostpreferably as a single continuous region.

Representative examples of polypeptide fragments of the receptor of thepresent invention include, for example, fragments comprising, oralternatively consisting of, from about amino acid number 1-20, 20-228,228-244 or 244-520 or the end of the coding region of SEQ ID NO: 2.

In another aspect of the present invention, differences as well assimilarities between sequences may comprise a preferred embodiment of aprotein or polypeptide of the present invention. Conserved domains orsequences may indicate that the conserved domain or sequence isindispensable for function. Differences in the amino acid composition ofproteins or polypeptides are also an important aspect of the presentinvention. For example, a difference in an amino acid at a singleposition may trigger an unwanted immunological response against aprotein or polypeptide. This may occur, for example, when the protein orpolypeptide is being used therapeutically. In addition, an amino aciddifference may affect the potency or the functionality of the proteinsor polypeptides of the present invention.

Representative examples of polypeptide or polypeptide fragments of theIFN-λs of the present invention include, for example, polypeptidescomprising, or alternatively consisting of:

R38-L47 of SEQ ID NO: 8, wherein Q49 may be any polar or charged aminoacid, for example K;F39-L47 of SEQ ID NO: 8, wherein Q49 may be any polar or charged aminoacid, for example K;S49-E58 of SEQ ID NO: 8, wherein S49 may be any amino acid having asmall side chain, i.e., G, A, S, C, or the like, preferably S or A andmost preferably S, R54 may be any charged amino acid, i.e., D, E, K orR, preferably R or K, and most preferably R, L57 may be any hydrophobicamino acid, i.e., A, V, F, P, M, I or L, preferably L or I, mostpreferably L;F50-E59 of SEQ ID NO: 8, wherein R54 may be any charged amino acid,i.e., D, E, K or R, preferably R or K, and most preferably R, L57 may beany hydrophobic amino acid, i.e., A, V, F, P, M, I or L, preferably L orI, most preferably L, E59 may be any charged amino acid, i.e., D, E, Kor R, preferably E or K, most preferably E;A90-K100 of SEQ ID NO: 8, wherein E92 may be any charged amino acid,i.e., D, E, K or R, preferably E or Q, and most preferably E, L95 may beany hydrophobic amino acid, i.e., A, V, F, P, M, I or L, preferably L orV, most preferably L;

W149-A156 of SEQ ID NO: 8; G163-T170 of SEQ ID NO: 8; N172-K182 of SEQID NO: 8;

An exact match for W77-L191 of SEQ ID NO: 8;An exact match for W149-L191 of SEQ ID NO: 8;An exact match for C34-T170 of SEQ ID NO: 8;An exact match for C34-T178 of SEQ ID NO: 8;An exact match for C34-T200 of SEQ ID NO: 8;An exact match for W77-T170 of SEQ ID NO: 8;An exact match for W77-T178 of SEQ ID NO: 8;An exact match for W77-T200 of SEQ ID NO: 8;An exact match for W149-T170 of SEQ ID NO: 8;An exact match for W149-T178 of SEQ ID NO: 8;An exact match for W149-T200 of SEQ ID NO: 8;C37-E61 of SEQ ID NO: 10, wherein Q48 may be any polar or charged aminoacid, for example D, E, K, R, S, T, Y, H, C, N, Q, W, preferably Q or K,most preferably, Q, R55 may be any charged amino acid, i.e., D, E, K orR, preferably R or K, most preferably R, L60 may be any hydrophobicamino acid, i.e., A, V, F, P, M, I or L, preferably L or I, mostpreferably L;C37-E62 of SEQ ID NO: 10, wherein Q48 may be any polar or charged aminoacid, for example D, E, K, R, S, T, Y, H, C, N, Q, W, preferably Q or K,most preferably, Q, R55 may be any charged amino acid, i.e., D, E, K orR, preferably R or K, most preferably R, L60 may be any hydrophobicamino acid, i.e., A, V, F, P, M, I or L, preferably L or I, mostpreferably L, E62 may be any charged amino acid, i.e., D, E, K or R,preferably E or K, most preferably E;W80-P91 of SEQ ID NO: 10, wherein R83 may be any charged amino acid,i.e., D, E, K or R, preferably R or K, most preferably R, R88 may be anycharged amino acid, i.e., D, E, K or R, preferably R or Q, mostpreferably R;W154-A161 of SEQ ID NO: 10, wherein Y156 may be any polar amino acid,i.e., S, T, Y, H, C, N, Q, W, preferably Y or H, most preferably Y;K164-T175 of SEQ ID NO: 10, wherein S166 may be any polar amino acid,i.e., S, T, Y, H, C, N, Q, W, preferably S or T, most preferably S;

N177-T183 of SEQ ID NO: 10;

An exact match for C37-T175 of SEQ ID NO: 10;An exact match for C37-T183 of SEQ ID NO: 10;An exact match for C37-V196 of SEQ ID NO: 10;An exact match for W80-T175 of SEQ ID NO: 10;An exact match for W80-T183 of SEQ ID NO: 10;An exact match for W80-V196 of SEQ ID NO: 10;An exact match for W154-T175 of SEQ ID NO: 10;An exact match for W154-T183 of SEQ ID NO: 10;An exact match for W154-V196 of SEQ ID NO: 10;C37-E61 of SEQ ID NO: 12, wherein Q48 may be any polar or charged aminoacid, for example D, E, K, R, S, T, Y, H, C, N, Q, W, preferably Q or K,most preferably, Q, R55 may be any charged amino acid, i.e., D, E, K orR, preferably R or K, most preferably R, L60 may be any hydrophobicamino acid, i.e., A, V, F, P, M, I or L, preferably L or I, mostpreferably L;C37-E62 of SEQ ID NO: 12, wherein Q48 may be any polar or charged aminoacid, for example D, E, K, R, S, T, Y, H, C, N, Q, W, preferably Q or K,most preferably, Q, R55 may be any charged amino acid, i.e., D, E, K orR, preferably R or K, most preferably R, L60 may be any hydrophobicamino acid, i.e., A, V, F, P, M, I or L, preferably L or I, mostpreferably L, E62 may be any charged amino acid, i.e., D, E, K or R,preferably E or K, most preferably E;W80-P91 of SEQ ID NO: 12, wherein R83 may be any charged amino acid,i.e., D, E, K or R, preferably R or K, most preferably R, R88 may be anycharged amino acid, i.e., D, E, K or R, preferably R or K, mostpreferably R;W154-A161 of SEQ ID NO: 12, wherein Y156 may be any polar amino acid,i.e., S, T, Y, H, C, N, Q, W, preferably Y or H, most preferably Y;K164-T175 of SEQ ID NO: 12, wherein S166 may be any polar amino acid,i.e., S, T, Y, H, C, N, Q, W, preferably S or T, most preferably S;

N177-T183 of SEQ ID NO: 12;

An exact match for C37-T175 of SEQ ID NO: 12;An exact match for C37-T183 of SEQ ID NO: 12;An exact match for C37-V196 of SEQ ID NO: 12;An exact match for W80-T175 of SEQ ID NO: 12;An exact match for W80-T183 of SEQ ID NO: 12;An exact match for W80-V196 of SEQ ID NO: 12;An exact match for W154-T175 of SEQ ID NO: 12;An exact match for W154-T183 of SEQ ID NO: 12;An exact match for W154-V196 of SEQ ID NO: 12.

In another embodiment the present invention, in any given polypeptide ofthe present invention, a specific amino acid is an exact match at aparticular position. These amino acids distinguish between the forms ofIFN-λ, and between allelic variations of a single form.

In another set of embodiments, the following positions, eachindependently or in combination, is preferred: position 2 of SEQ ID NO:8 is not T, most preferably it is A, position 4 of SEQ ID NO: 8 is notD, most preferably it is A, position 5 of SEQ ID NO: 8 is not C, mostpreferably it is W, position 6 of SEQ ID NO: 8 is not M, most preferablyit is T, position 7 of SEQ ID NO: 8 is not P, most preferably it is V,position 11 of SEQ ID NO: 8 is not L, most preferably it is T, position13 of SEQ ID NO: 8 is not A, most preferably it is V, position 14 of SEQID NO: 8 is not A, most preferably it is L, position 15 of SEQ ID NO: 8is not V, most preferably it is G, position 17 of SEQ ID NO: 8 is not T,most preferably it is A, position 19 of SEQ ID NO: 8 is not T, mostpreferably it is A, position 21 of SEQ ID NO: 8 is not A, mostpreferably it is P, position 24 of SEQ ID NO: 8 is not V, mostpreferably it is T, position 25 of SEQ ID NO: 8 is not A, mostpreferably it is S, position 26 of SEQ ID NO: 8 is not R, mostpreferably it is K, position 27 of SEQ ID NO: 8 is not A, mostpreferably it is P, position 28 of SEQ ID NO: 8 is not L, mostpreferably it is T, position 29 of SEQ ID NO: 8 is not T, mostpreferably it is P, position 30 of SEQ ID NO: 8 is not D, mostpreferably it is T, position 31 of SEQ ID NO: 8 is not A, mostpreferably it is G, position 32 of SEQ ID NO: 8 is not R, mostpreferably it is K, position of SEQ ID NO: 837 is not A, most preferablyit is G, position 38 of SEQ ID NO: 8 is not Q, most preferably it is R,position 48 of SEQ ID NO: 8 is not Q, most preferably it is A, position52 of SEQ ID NO: 8 is not R, most preferably it is K, position 54 of SEQID NO: 8 is not K, most preferably it is R, position 62 of SEQ ID NO: 8is not L, most preferably it is K, position 65 of SEQ ID NO: 8 is not D,most preferably it is N, position 66 of SEQ ID NO: 8 is not T or C, mostpreferably it is W, position 67 of SEQ ID NO: 8 is not R or K, mostpreferably it is S, position 69 of SEQ ID NO: 8 is not R or H, mostpreferably it is S, position 71 of SEQ ID NO: 8 is not R, mostpreferably it is P, position 72 of SEQ ID NO: 8 is not L, mostpreferably it is V, position 75 of SEQ ID NO: 8 is not R, mostpreferably it is G, position 76 of SEQ ID NO: 8 is not T, mostpreferably it is N, position 81 of SEQ ID NO: 8 is not Q, mostpreferably it is L, position 89 of SEQ ID NO: 8 is not M, mostpreferably it is V, position 105 of SEQ ID NO: 8 is not T or S, mostpreferably it is A, position 106 of SEQ ID NO: 8 most preferably is A,position 107 of SEQ ID NO: 8 most preferably is G, position 111 of SEQID NO: 8 is not V or G, most preferably it is E, position 128 of SEQ IDNO: 8 is not F, most preferably it is L, position 129 of SEQ ID NO: 8 isnot R, most preferably it is Q, position 142 of SEQ ID NO: 8 is not A orT, most preferably it is P, position 151 of SEQ ID NO: 8 is not Y, mostpreferably it is H, position 162 of SEQ ID NO: 8 is not P, mostpreferably it is A, position 182 of SEQ ID NO: 8 is not N, mostpreferably it is K, position 183 of SEQ ID NO: 8 is not C, mostpreferably it is Y, position 186 of SEQ ID NO: 8 is not S, mostpreferably it is D, position 188 of SEQ ID NO: 8 is not D, mostpreferably it is N, position 191 of SEQ ID NO: 8 is not V, mostpreferably it is L, position 192 of SEQ ID NO: 8 most preferably is R,position 193 of SEQ ID NO: 8 most preferably is T, position 194 of SEQID NO: 8 most preferably is S, position 195 of SEQ ID NO: 8 mostpreferably is T, position 196 of SEQ ID NO: 8 most preferably is H,position 197 of SEQ ID NO: 8 most preferably is P, position 198 of SEQID NO: 8 most preferably is E, position 199 of SEQ ID NO: 8 mostpreferably is S, position 200 of SEQ ID NO: 8 most preferably is T.

In another set of embodiments, the following positions, eachindependently or in combination, is preferred: position 2 of SEQ ID NO:10 is not A, most preferably it is T; position 3 of SEQ ID NO: 10 is notA, most preferably it is G; position 4 of SEQ ID NO: 10 is not A, mostpreferably it is D; position 5 of SEQ ID NO: 10 is not W, mostpreferably it is C; position 6 of SEQ ID NO: 10 is not M, mostpreferably it is T; position 7 of SEQ ID NO: 10 is not V, mostpreferably it is P; position 11 of SEQ ID NO: 10 is not T, mostpreferably it is L; position 12 of SEQ ID NO: 10 is not L, mostpreferably it is M; position 13 of SEQ ID NO: 10 is not V, mostpreferably it is A; position 14 of SEQ ID NO: 10 is not L, mostpreferably it is A; position 15 of SEQ ID NO: 10 is not G, mostpreferably it is V; position 17 of SEQ ID NO: 10 is not A, mostpreferably it is T; position 19 of SEQ ID NO: 10 is not A, mostpreferably it is T; position 21 of SEQ ID NO: 10 is not P, mostpreferably it is A; position 24 of SEQ ID NO: 10 is not T, mostpreferably it is V; position 25 of SEQ ID NO: 10 is not S, mostpreferably it is A; position 26 of SEQ ID NO: 10 is not K, mostpreferably it is R; position 27 of SEQ ID NO: 10 most preferably is L;position 28 of SEQ ID NO: 10 is not R, most preferably it is H; position29 of SEQ ID NO: 10 most preferably is G; position 30 of SEQ ID NO: 10is not P, most preferably it is A; position 31 of SEQ ID NO: 10 is notT, most preferably it is L; position 33 of SEQ ID NO: 10 is not T, mostpreferably it is D; position 34 of SEQ ID NO: 10 is not G, mostpreferably it is A; position 35 of SEQ ID NO: 10 is not K, mostpreferably it is R; position 40 of SEQ ID NO: 10 is not G, mostpreferably it is A; position 41 of SEQ ID NO: 10 is not R, mostpreferably it is Q; position 51 of SEQ ID NO: 10 is not A, mostpreferably it is Q; position 52 of SEQ ID NO: 10 is not S, mostpreferably it is A; position 55 of SEQ ID NO: 10 is not K, mostpreferably it is R; position 57 of SEQ ID NO: 10 is not R, mostpreferably it is K; position 65 of SEQ ID NO: 10 is not K, mostpreferably it is L; position 68 of SEQ ID NO: 10 is not N, mostpreferably it is D; position 69 of SEQ ID NO: 10 is not W, mostpreferably it is C; position 70 of SEQ ID NO: 10 is not S or K, mostpreferably it is R; position 72 of SEQ ID NO: 10 is not R or S, mostpreferably it is H; position 74 of SEQ ID NO: 10 is not P, mostpreferably it is R; position 75 of SEQ ID NO: 10 is not V, mostpreferably it is L; position 78 of SEQ ID NO: 10 is not G, mostpreferably it is R; position 79 of SEQ ID NO: 10 is not N, mostpreferably it is T; position 84 of SEQ ID NO: 10 is not L, mostpreferably it is Q; position 92 of SEQ ID NO: 10 is not V, mostpreferably it is M; position 108 of SEQ ID NO: 10 is not S or A, mostpreferably it is T; position 110 of SEQ ID NO: 10 most preferably is D;position 111 of SEQ ID NO: 10 most preferably is T; position 112 of SEQID NO: 10 is not G, most preferably it is D; position 116 of SEQ ID NO:10 is not G or E, most preferably it is V; position 133 of SEQ ID NO: 10is not L, most preferably it is F; position 134 of SEQ ID NO: 10 is notQ, most preferably it is R; position 147 of SEQ ID NO: 10 is not T or P,most preferably it is A; position 156 of SEQ ID NO: 10 is not H, mostpreferably it is Y; position 167 of SEQ ID NO: 10 is not A, mostpreferably it is P; position 187 of SEQ ID NO: 10 is not K, mostpreferably it is N; position 188 of SEQ ID NO: 10 is not Y, mostpreferably it is C; position 191 of SEQ ID NO: 10 is not D, mostpreferably it is S; position 193 of SEQ ID NO: 10 is not N, mostpreferably it is D; position 196 of SEQ ID NO: 10 is not L, mostpreferably it is V.

In another set of embodiments, the following positions, eachindependently or in combination, is preferred: position 2 of SEQ ID NO:12 is not A, most preferably it is T; position 3 of SEQ ID NO: 12 is notA, most preferably it is G; position 4 of SEQ ID NO: 12 is not A, mostpreferably it is D; position 5 of SEQ ID NO: 12 is not W, mostpreferably it is C; position 6 of SEQ ID NO: 12 is not T, mostpreferably it is M; position 7 of SEQ ID NO: 12 is not V, mostpreferably it is P; position 11 of SEQ ID NO: 12 is not T, mostpreferably it is L; position 12 of SEQ ID NO: 12 is not L, mostpreferably it is M; position 13 of SEQ ID NO: 12 is not V, mostpreferably it is A; position 14 of SEQ ID NO: 12 is not L, mostpreferably it is A; position 15 of SEQ ID NO: 12 is not G, mostpreferably it is V; position 17 of SEQ ID NO: 12 is not A, mostpreferably it is T; position 19 of SEQ ID NO: 12 is not A, mostpreferably it is T; position 21 of SEQ ID NO: 12 is not P, mostpreferably it is A; position 24 of SEQ ID NO: 12 is not T, mostpreferably it is V; position 25 of SEQ ID NO: 12 is not S, mostpreferably it is A; position 26 of SEQ ID NO: 12 is not K, mostpreferably it is R; position 27 of SEQ ID NO: 12 most preferably is L;position 28 of SEQ ID NO: 12 is not H, most preferably is R; position 29of SEQ ID NO: 12 most preferably is G; position 30 of SEQ ID NO: 12 isnot P, most preferably it is A; position 31 of SEQ ID NO: 12 is not T,most preferably it is L; position 33 of SEQ ID NO: 12 is not T, mostpreferably it is D; position 34 of SEQ ID NO: 12 is not G, mostpreferably it is A; position 35 of SEQ ID NO: 12 is not K, mostpreferably it is R; position 40 of SEQ ID NO: 12 is not G, mostpreferably it is A; position 41 of SEQ ID NO: 12 is not R, mostpreferably it is Q; position 51 of SEQ ID NO: 12 is not A, mostpreferably it is Q; position 52 of SEQ ID NO: 12 is not S, mostpreferably it is A; position 55 of SEQ ID NO: 12 is not K, mostpreferably it is R; position 57 of SEQ ID NO: 12 is not R, mostpreferably it is K; position 65 of SEQ ID NO: 12 is not K, mostpreferably it is L; position 68 of SEQ ID NO: 12 is not N, mostpreferably it is D; position 69 of SEQ ID NO: 12 is not W, mostpreferably it is C; position 70 of SEQ ID NO: 12 is not S or R, mostpreferably it is K; position 72 of SEQ ID NO: 12 is not H or S, mostpreferably it is R; position 74 of SEQ ID NO: 12 is not P, mostpreferably it is R; position 75 of SEQ ID NO: 12 is not V, mostpreferably it is L; position 78 of SEQ ID NO: 12 is not G, mostpreferably it is R; position 79 of SEQ ID NO: 12 is not N, mostpreferably it is T; position 84 of SEQ ID NO: 12 is not L, mostpreferably it is Q; position 92 of SEQ ID NO: 12 is not M, mostpreferably it is V; position 108 of SEQ ID NO: 12 is not T or A, mostpreferably it is S; position 110 of SEQ ID NO: 12 most preferably is D;position 111 of SEQ ID NO: 12 most preferably is T; position 112 of SEQID NO: 12 is not G, most preferably it is D; position 116 of SEQ ID NO:12 is not V or E, most preferably it is G; position 133 of SEQ ID NO: 12is not F, most preferably it is L; position 134 of SEQ ID NO: 12 is notQ, most preferably it is R; position 147 of SEQ ID NO: 12 is not A or P,most preferably it is T; position 156 of SEQ ID NO: 12 is not H, mostpreferably it is Y; position 167 of SEQ ID NO: 12 is not A, mostpreferably it is P; position 187 of SEQ ID NO: 12 is not K, mostpreferably it is N; position 188 of SEQ ID NO: 12 is not Y, mostpreferably it is C; position 191 of SEQ ID NO: 12 is not D, mostpreferably it is S; position 193 of SEQ ID NO: 10 is not N, mostpreferably it is D; position 196 of SEQ ID NO: 12 is not L, mostpreferably it is V.

In another set of embodiments, the positions described supra, may bereversely altered such that one IFN-λ type may more closely resembleanother type, or alternatively, an allele.

Polypeptide fragments can be about 20, 30, 40, 50, 60, 70, 80, 90, 100,110, 120, 130, 140, or 150 amino acids in length. In this context“about” includes the particularly recited ranges or values, and rangesor values larger or smaller by several (5, 4, 3, 2, or 1) amino acids,at either extreme or at both extremes. Polynucleotides encoding thesepolypeptides are also encompassed by the invention.

For many proteins, including the extracellular domain of membraneassociated protein or the mature form(s) of a secreted protein, it isknown in the art that one or more amino acids may be deleted from theN-terminus or C-terminus without substantial loss of biologicalfunction. However, even if deletion of one or more amino acids from theN-terminus or C-terminus of a protein results in modification of loss ofone or more biological functions of the protein, other biologicalactivities may still be retained. Thus, the ability of the shortenedprotein to induce and/or bind to antibodies which recognize the completeprotein generally will be retained when less than the majority of theresidues of the complete protein are removed from the N-terminus.Whether a particular polypeptide lacking N-terminal residues of acomplete protein retains such immunologic activities can readily bedetermined by routine methods described herein and otherwise known inthe art.

Interferon gamma has been reported to show up to ten times higheractivities by deleting 8-10 amino acid residues from the carboxyterminus of the protein (Dobeli et al., J. Biotechnology 7:199-216(1988). In the present case deletions of C-terminal amino acids of theIFN-λ polypeptides of the present invention may retain some biologicalactivity, such as antiviral activity, modulation of MHC, apoptosiseliciting activity expression or binding to the receptor complexcomprising IFN-λR1.

However, even if deletion of one or more amino acids from the C-terminusof a protein results in modification of loss of one or more biologicalfunctions of the protein, other biological activities may still beretained. Thus, the ability of the shortened protein to induce and/orbind to antibodies which recognize the complete protein generally willbe retained when less than the majority of the residues of the completeprotein are removed from the C-terminus. Whether a particularpolypeptide lacking C-terminal residues of a complete protein retainssuch immunologic activities can readily be determined by routine methodsdescribed herein and otherwise known in the art.

The present application is also directed to nucleic acid moleculescomprising, or alternatively, consisting of, a polynucleotide sequenceencoding the polypeptide described above. The present invention alsoencompasses the above polynucleotide sequences fused to a heterologouspolynucleotide sequence.

The present application is also directed to nucleic acid moleculescomprising, or alternatively, consisting of, a polynucleotide sequenceat least 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to thepolynucleotide sequence encoding the IFN-λ polypeptides described above.The present invention also encompasses the above polynucleotidesequences fused to a heterologous polynucleotide sequence.

Preferably, the polynucleotide fragments of the invention encode apolypeptide which demonstrates an IFN-λ functional activity. By apolypeptide demonstrating an IFN-λ “functional activity” is meant, apolypeptide capable of displaying one or more known functionalactivities associated with a full-length (complete) an IFN-λ protein.Such functional activities include, but are not limited to, biologicalactivity (e.g., anti-viral activity, modulation of MHC expression,immunomodulatory activity, regulator of tumor cell growth, ability toinduce apoptosis), antigenicity (ability to bind, or compete with anIFNλ polypeptide for binding, to an anti-IFN-λ antibody), immunogenicity(ability to generate antibody which binds to an IFN-λ polypeptide),ability to bind to and/or activate the receptor complex comprisingIFN-λR1, and the like.

The functional activity of the polypeptides of the present invention,and fragments, variants derivatives, and analogs thereof, can be assayedby various methods.

For example, in one embodiment where one is assaying for the ability tobind or compete with a full-length IFN-λ polypeptide for binding toanti-IFNλ antibody, various immunoassays known in the art can be used,including but not limited to, competitive and non-competitive assaysystems using techniques such as radioimmunoassays, ELISA (enzyme linkedimmunosorbent assay), “sandwich” immunoassays, immunoradiometric assays,gel diffusion precipitation reactions, immunodiffusion assays, in situimmunoassays (using colloidal gold, enzyme or radioisotope labels, forexample), western blots, precipitation reactions, agglutination assays(e.g., gel agglutination assays, hemagglutination assays), complementfixation assays, immunofluorescence assays, protein A assays, andimmunoelectrophoresis assays, etc. In one embodiment, antibody bindingis detected by detecting a label on the primary antibody. In anotherembodiment, the primary antibody is detected by detecting binding of asecondary antibody or reagent to the primary antibody. In a furtherembodiment, the secondary antibody is labeled. Many means are known inthe art for detecting binding in an immunoassay and are within the scopeof the present invention.

In another embodiment, physiological correlates of IFN-λ binding to itssubstrates (signal transduction) can be assayed. In addition, assaysdescribed herein and otherwise known in the art may routinely be appliedto measure the ability of IFN-λ polypeptides and fragments, variantsderivatives and analogs thereof to elicit IFN-λ related biologicalactivity (either in vitro or in vivo). Other methods will be known tothe skilled artisan and are within the scope of the invention.

It also will be recognized by one of ordinary skill in the art that someamino acid sequences of the IFN-λ polypeptides can be varied withoutsignificant effect on the structure or function of the protein.

Thus, the invention further includes variations of the IFN-λ polypeptidewhich show substantial IFN-λ polypeptide activity or which includeregions of IFN-λ protein such as the protein portions discussed below.Such mutants include deletions, insertions, inversions, repeats, splicevariants and type substitutions selected according to general rulesknown in the art so as have little effect on activity. For example,guidance concerning how to make phenotypically silent amino acidsubstitutions is provided in Bowie, J. U. et al., “Deciphering theMessage in Protein Sequences: Tolerance to Amino Acid Substitutions,”Science 247:1306-1310 (1990), wherein the authors indicate that thereare two main approaches for studying the tolerance of an amino acidsequence to change. The first method relies on the process of evolution,in which mutations are either accepted or rejected by natural selection.The second approach uses genetic engineering to introduce amino acidchanges at specific positions of a cloned gene and selections or screensto identify sequences that maintain functionality.

Thus, the fragment, derivative or analog of the polypeptides of SEQ IDNO: 2, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 may be(i) one in which one or more of the amino acid residues are substitutedwith a conserved or non-conserved amino acid residue (preferably aconserved amino acid residue) and such substituted amino acid residuemay or may not be one encoded by the genetic code, or (ii) one in whichone or more of the amino acid residues includes a substituent group, or(iii) one in which the polypeptide is fused with another compound, suchas a compound to increase the half-life of the polypeptide (for example,polyethylene glycol, or albumin), or (iv) one in which the additionalamino acids are fused to the above form of the polypeptide, such as anIgG Fc fusion region peptide or leader or secretory sequence or asequence which is employed for purification of the above form of thepolypeptide or a proprotein sequence. Such fragments, derivatives andanalogs are deemed to be within the scope of those skilled in the artfrom the teachings herein.

Thus, the polypeptides of the present invention may include one or moreamino acid substitutions, deletions or additions, either from naturalmutations or human manipulation. As indicated, changes are preferably ofa minor nature, such as conservative amino acid substitutions that donot significantly affect the folding or activity of the protein.Additional variant polypeptides of the present invention includeexpression variants that enhance secretion or increase the biologicalactivity of the polypeptide of the present invention.

Amino acids in the polypeptides of the present invention that areessential for function can be identified by methods known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244:1081-1085 (1989).) The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as receptor binding, in vitro proliferative activity orinterferon receptor activation.

Of special interest are substitutions of charged amino acids with othercharged or neutral amino acids which may produce proteins with highlydesirable improved characteristics, such as less aggregation.Aggregation may not only reduce activity but also be problematic whenpreparing pharmaceutical formulations, because aggregates can beimmunogenic (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967);Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev.Therapeutic Drug Carrier Systems 10:307-377 (1993).)

Replacement of amino acids can also change the selectivity of thebinding of a ligand to cell surface receptors. For example, Ostade etal., Nature 361:266-268 (1993) describes certain mutations resulting inselective binding of TNF-.alpha. to only one of the two known types ofTNF receptors. Sites that are critical for ligand-receptor binding canalso be determined by structural analysis such as crystallization,nuclear magnetic resonance or photoaffinity labeling (Smith et al., J.Mol. Biol. 224:899-904 (1992) and de Vos et al. Science 255:306-312(1992)).

The polypeptides of the present invention are preferably provided in anisolated form, and preferably are substantially purified. Thepolypeptides may be produced, isolated and purified by any effectivemanner known in the art.

For example, recombinantly produced versions of IFN-λs and solubleCRF2-12 polypeptides can be substantially purified by the one-stepmethod described in Smith and Johnson, Gene 67:31-40 (1988).Polypeptides of the invention also can be purified from natural orrecombinant sources using specific antibodies of the invention,described infra, in methods which are well known in the art of proteinpurification. By “% similarity” for two polypeptides is intended, forexample, a similarity score produced by comparing the amino acidsequences of the two polypeptides. This may be done, for example, usingthe Bestfit program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711) and the default settings for determiningsimilarity. Bestfit uses the local homology algorithm of Smith andWaterman (Advances in Applied Mathematics 2:482-489, 1981) to find thebest segment of similarity between two sequences.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a query amino acid sequence of a polypeptide of theinvention it is intended that the amino acid sequence of the subjectpolypeptide is identical to the query sequence except that the subjectpolypeptide sequence may include up to five amino acid alterations pereach 100 amino acids of the query amino acid sequence of the presentinvention. In other words, to obtain a polypeptide having an amino acidsequence at least 95% identical to a query amino acid sequence, up to 5%of the amino acid residues in the subject sequence may be inserted,deleted, or substituted with another amino acid, or a number of aminoacids up to 5% of the total amino acid residues in the query sequencemay be inserted into the subject sequence. These alterations of thesubject sequence may occur at the amino or carboxy terminal positions ofthe reference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

As described in detail below, the polypeptides of the present inventioncan also be used to raise polyclonal and monoclonal antibodies, whichare useful in assays for detecting IFN-λ protein expression and CRF2-12protein expression, as described below, or as agonists and antagonistscapable of enhancing or inhibiting IFN-λ protein function and CRF2-12receptor or receptor complex function. Further, such polypeptides can beused in the yeast two-hybrid system to “capture” IFN-λ binding proteins,and proteins that bind to the intracellular, extracellular or thetransmembrane domain of the CRF2-12 receptor or the receptor complex.Captured proteins are candidate agonists and antagonists according tothe present invention. The yeast two hybrid system is described inFields and Song, Nature 340:245-246 (1989).

Among the especially preferred polypeptide fragments of the inventionare fragments characterized by structural or functional attributes ofthe proteins and polypeptides of the present invention. Such fragmentsinclude amino acid residues that comprise alpha-helix and alpha-helixforming regions (“alpha-regions”), beta-sheet and beta-sheet-formingregions (“beta-regions”), turn and turn-forming regions(“turn-regions”), coil and coil-forming regions (“coil-regions”),hydrophilic regions, hydrophobic regions, alpha amphipathic regions,beta amphipathic regions, surface forming regions, and high antigenicindex regions (i.e., containing four or more contiguous amino acidshaving an antigenic index of greater than or equal to 1.5, as identifiedusing the default parameters of the Jameson-Wolf program).

Other preferred regions include: Garnier-Robson predicted alpha-regions,beta-regions, turn-regions, and coil-regions; Chou-Fasman predictedalpha-regions, beta-regions, and turn-regions; Kyte-Doolittle predictedhydrophilic regions; Eisenberg alpha and beta amphipathic regions; Eminisurface-forming regions; Karplus-Schulz predicted flexible regions; andJameson-Wolf high antigenic index regions, as predicted using thedefault parameters of these computer programs. Polynucleotides encodingthese polypeptides are also encompassed by the invention.

It has been reported that Interferon alpha possesses a wide variety ofantiviral, anti-proliferative and immunomodulative biologicalactivities. These multiple activities of interferon are thought to bemediated through interaction with specific cell-surface receptors. Theinterferon receptor generally is believed to consists of more than oneindividual polypeptide component and different parts of the interferonmolecule are thought to contribute to certain interferon activities viainteraction with distinct chains of the interferon receptor complex.Wang et al., J. Immunol. 152:705-715; Uze et al. J. Mol. Biol.243:245-257 (1994). The structure-functional organization of the type 1interferon molecule, the interferon receptor complex, and the role ofdistinct receptor chains in signal transduction has been analyzed.Danilkovitch et al. Hybridoma 16:69-75 (1997); Pontzer et al. J.Interferon Res 14: 133-141 (1994); Danilkovich et al., ImmunologyLetters 31:15-20 (1991). Polypeptide fragments of the present invention,therefore, may be used to mediate antiviral, antiproliferative,apoptotic and immunomodulative biological activities.

Polypeptide fragments from the C-terminus of Interferon-alpha2 exhibitantiproliferative activity on normal human peripheral blood lymphocytes.Epitopes involving amino acids 124-144 of the Interferon-alpha2 moleculeare thought to be responsible for receptor binding and for themanifestation of interferon antiproliferative properties. Danilkovich etal. Immunology Letters, supra. Polypeptide fragments from thecarboxy-terminal region of Interferon-tau are thought to be involved inthe antiviral activity by a mechanism and specificity shared by alphaInterferons. Pontzer et al. Proc. Natl. Acad. Sci. USA 87:5945-5949(1990). Accordingly, polypeptide fragments of the present invention maybe used as physiological regulators of tumor cell growth,anti-proliferative activity, anti-viral activity apoptotic activity andimmunomodulatory activity.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NOS: 1-12 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention.

The present invention encompasses polypeptides comprising, oralternatively consisting of, an epitope of the polypeptide having anamino acid sequence of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 8, SEQ IDNO: 10, SEQ ID NO: 12.

The present invention further encompasses polynucleotide sequencesencoding an epitope of a polypeptide sequence of the invention,polynucleotide sequences of the complementary strand of a polynucleotidesequence encoding an epitope of the invention, and polynucleotidesequences which hybridize to the complementary strand under stringenthybridization conditions or lower stringency hybridization conditionsdefined supra.

The term “epitopes,” as used herein, refers to portions of a polypeptidehaving antigenic or immunogenic activity in an animal, preferably amammal, and most preferably in a human, mouse, rabbit or rat. In apreferred embodiment, the present invention encompasses a polypeptidecomprising an epitope, as well as the polynucleotide encoding thispolypeptide. An “immunogenic epitope,” as used herein, is defined as aportion of a protein that elicits an antibody response in an animal, asdetermined by any method known in the art, for example, by the methodsfor generating antibodies described infra. (See, for example, Geysen etal., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983)). The term“antigenic epitope,” as used herein, is defined as a portion of aprotein to which an antibody can immunospecifically bind its antigen asdetermined by any method well known in the art, for example, by theimmunoassays described herein. Immunospecific binding excludesnon-specific binding but does not necessarily exclude cross-reactivitywith other antigens. Antigenic epitopes need not necessarily beimmunogenic.

Fragments which function as epitopes may be produced by any conventionalmeans. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135(1985), further described in U.S. Pat. No. 4,631,211).

In the present invention, antigenic epitopes preferably contain asequence of at least 4, at least 5, at least 6, at least 7, morepreferably at least 8, at least 9, at least 10, at least at least 12, atleast 13, at least 14, at least 15, at least 20, at least 25, at least30, at least 11, least 50, and, most preferably, between about 15 toabout 30 amino acids. Preferred polypeptides comprising immunogenic orantigenic epitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length.Additional non-exclusive preferred antigenic epitopes include theantigenic epitopes disclosed herein, as well as portions thereof.Antigenic epitopes are useful, for example, to raise antibodies,including monoclonal antibodies, that specifically bind the epitope.Preferred antigenic epitopes include the antigenic epitopes disclosedherein, as well as any combination of two, three, four, five or more ofthese antigenic epitopes. Antigenic epitopes can be used as the targetmolecules in immunoassays. (See, for instance, Wilson et al., Cell37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983)).

Similarly, immunogenic epitopes can be used, for example, to induceantibodies according to methods well known in the art. (See, forinstance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al.,Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol.66:2347-2354 (1985). Preferred immunogenic epitopes include theimmunogenic epitopes disclosed herein, as well as any combination oftwo, three, four, five or more of these immunogenic epitopes. Thepolypeptides comprising one or more immunogenic epitopes may bepresented for eliciting an antibody response together with a carrierprotein, such as an albumin, to an animal system (such as rabbit ormouse), or, if the polypeptide is of sufficient length (at least about25 amino acids), the polypeptide may be presented without a carrier.However, immunogenic epitopes comprising as few as 8 to 10 amino acidshave been shown to be sufficient to raise antibodies capable of bindingto, at the very least, linear epitopes in a denatured polypeptide (e.g.,in Western blotting).

Epitope-bearing polypeptides of the present invention may be used toinduce antibodies according to any effective method, including in vivoimmunization, in vitro immunization, and phage display methods. See,e.g., Sutcliffe et al., supra; Wilson et al., supra, and Bittle et al.,J. Gen. Virol., 66:2347-2354 (1985). If in vivo immunization is used,animals may be immunized with free peptide. Anti-peptide antibody titermay also be boosted by coupling the peptide to a macromolecular carrier,such as keyhole limpet hemacyanin (KLH) or tetanus toxoid.

Peptides containing cysteine residues may be coupled to a carrier usinga linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),while other peptides may be coupled to carriers using a more generallinking agent such as glutaraldehyde. Animals such as rabbits, rats andmice are immunized with either free or carrier-coupled peptides, forinstance, by intraperitoneal and/or intradermal injection of emulsionscontaining about 100 μg of peptide or carrier protein and Freund'sadjuvant or any other adjuvant known for stimulating an immune response.Several booster injections may be needed, for instance, at intervals ofabout two weeks, to provide a useful titer of anti-peptide antibodywhich can be detected, for example, by ELISA assay using free peptideadsorbed to a solid surface. The titer of anti-peptide antibodies inserum from an immunized animal may be increased by any effective method.For example, by selection of anti-peptide antibodies by adsorption tothe peptide on a solid support and elution of the selected antibodiesaccording to methods well known in the art. The polypeptides of thepresent invention (e.g., those comprising an immunogenic or antigenicepitope) can be fused to heterologous polypeptide sequences. Forexample, the polypeptides of the present invention (including fragmentsor variants thereof) may be fused with the constant domain ofimmunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CH1, CH2,CH3, or any combination thereof and portions thereof) resulting inchimeric polypeptides. By way of another non-limiting example,polypeptides and/or antibodies of the present invention (includingfragments or variants thereof) may be fused with albumin (including butnot limited to recombinant human serum albumin or fragments or variantsthereof (see, e.g., U.S. Pat. No. 5,876,969, issued Mar. 2, 1999, EPPatent 0 413 622, and U.S. Pat. No. 5,766,883, issued Jun. 16, 1998,herein incorporated by reference in their entirety)).

Such fusion proteins as those described above may facilitatepurification and may increase half-life in vivo. This has been shown forchimeric proteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins. See, e.g., EP 394,827;Traunecker et al., Nature, 331:84-86 (1988).

Additional fusion proteins of the invention may be generated through thetechniques of gene-shuffling, motif-shuffling, exon-shuffling, and/orcodon-shuffling (collectively referred to as “DNA shuffling”). DNAshuffling may be employed to modulate the activities of polypeptides ofthe invention, such methods can be used to generate polypeptides withaltered activity, as well as agonists and antagonists of thepolypeptides. See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238;5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. OpinionBiotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol. 16(2):76-82(1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzoand Blasco, Biotechniques 24(2):308-13 (1998) (each of these patents andpublications are hereby incorporated by reference in its entirety).

Recombinant and Synthetic Production of Polypeptides of the Invention

The present invention also relates to vectors which include the isolatedDNA molecules of the present invention, host cells which are geneticallyengineered with the recombinant vectors, and the production ofpolypeptides or fragments of polypeptides of the present invention byrecombinant and synthetic techniques. The vector may be any effectivevector. These include, for example, a phage, plasmid, viral orretroviral vector. Retroviral vectors may be replication competent orreplication defective. In the latter case, viral propagation generallywill occur only in complementing host cells.

The polynucleotides of the present invention, or encoding thepolypeptides or polypeptide fragments of the present invention may bejoined to a vector containing a selectable marker for propagation in ahost. Generally, a plasmid vector is introduced in a precipitate, suchas a calcium phosphate precipitate, or in a complex with a chargedlipid. If the vector is a virus, it may be packaged in vitro using anappropriate packaging cell line and then transduced into host cells.

The DNA insert should be operatively linked to an appropriate promoter.Any effective promoter may be used. These include the phage lambda PLpromoter, the E. coli lac, trp, phoA and tac promoters, the SV40 earlyand late promoters and promoters of retroviral LTRs, to name a few.Other suitable promoters will be known to the skilled artisan. Theexpression constructs preferably also contain sites for transcriptioninitiation, termination and, in the transcribed region, a ribosomebinding site for translation in an effective position with respect tothe DNA insert of the invention. The coding portion of the transcriptsexpressed by the constructs will preferably include a translationinitiating codon at the beginning and a termination codon (UAA, UGA orUAG) appropriately positioned at the end of the polypeptide to betranslated.

As indicated, the expression vectors will preferably include at leastone selectable marker. Any effective marker may be used. Such markersinclude dihydrofolate reductase, G418 or neomycin resistance foreukaryotic cell culture and tetracycline, kanamycin or ampicillinresistance genes for culturing in E. coli and other bacteria.Representative examples of appropriate hosts include, but are notlimited to, bacterial cells, such as E. coli, Streptomyces andSalmonella typhimurium cells; fungal cells, such as yeast cells (e.g.,Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No.201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells;animal cells such as CHO, COS, 293 and Bowes melanoma cells; and plantcells. Appropriate culture mediums and conditions for theabove-described host cells are known in the art.

Among vectors preferred for use in bacteria include pQE70, pQE60 andpQE-9, available from QIAGEN, Inc., supra; pBS vectors, Phagescriptvectors, pBluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, availablefrom Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3,pDR540, pRIT5 available from Pharmacia Biotech, Inc. Among preferredeukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG availablefrom Stratagene; and pSVK3, pBPV, pMSG and pSVL available fromPharmacia. Preferred expression vectors for use in yeast systemsinclude, but are not limited to pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ,pGAPZ, pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K,and PAO815 (all available from Invitrogen, Carlbad, Calif.). Othersuitable vectors will be readily apparent to the skilled artisan.

Introduction of the construct into the host cell can be effected by anyeffective means, including naked nucleic acid, use of a gene gun,calcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986). It is specifically contemplated that polypeptides orpolypeptide fragments of the present invention may be expressed by ahost cell lacking a recombinant vector.

The polypeptide may be expressed in a modified form, such as a fusionprotein, and may include not only secretion signals, but also additionalheterologous functional regions. For instance, a region of additionalamino acids, particularly charged amino acids, may be added to theN-terminus of the polypeptide to improve stability and persistence inthe host cell, during purification, or during subsequent handling andstorage. Also, peptide moieties may be added to the polypeptide tofacilitate purification. Such regions may be removed prior to finalpreparation of the polypeptide. The addition of peptide moieties topolypeptides to engender secretion or excretion, to improve stabilityand to facilitate purification, among others, are familiar and routinetechniques in the art. A preferred fusion protein comprises aheterologous region from immunoglobulin that is useful to stabilize andpurify proteins. For example, EP-A-O 464 533 (Canadian counterpart2045869) discloses fusion proteins comprising various portions ofconstant region of immunoglobulin molecules together with another humanprotein or part thereof. In many cases, the Fc part in a fusion proteinis thoroughly advantageous for use in therapy and diagnosis and thusresults, for example, in improved pharmacokinetic properties (EP-A 0232262). On the other hand, for some uses it would be desirable to be ableto delete the Fc part after the fusion protein has been expressed,detected and purified in the advantageous manner described. This is thecase when Fc portion proves to be a hindrance to use in therapy anddiagnosis, for example when the fusion protein is to be used as antigenfor immunizations. In drug discovery, for example, human proteins, suchas hIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. See,D. Bennett et al., J. Molecular Recognition 8:52-58 (1995) and K.Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).

The polypeptide and polypeptide fragments of the present invention maybe recovered and purified from recombinant cell cultures in anyeffective manner. For example, ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. For example, see Lin et al., “Purification ofRecombinant Human Interferon Beta expressed in E. coli” Methods inEnzymology 119: 183-192 (1986), which is hereby incorporated byreference in its entirety. Most preferably, high performance liquidchromatography (“HPLC”) is employed for purification.

Further methods that may be used for production and isolation of thepolypeptides of the present invention are disclosed in U.S. Pat. No.6,433,145.

Polypeptides of the present invention include: products purified fromnatural sources, including bodily fluids, tissues and cells, whetherdirectly isolated or cultured; products of chemical syntheticprocedures; and products produced by recombinant techniques from aprokaryotic or eukaryotic host, including, for example, bacterial,yeast, higher plant, insect and mammalian cells. Depending upon the hostemployed in a recombinant production procedure, the polypeptides of thepresent invention may be glycosylated or may be non-glycosylated. Inaddition, polypeptides of the invention may also include an initialmodified methionine residue, in some cases as a result of host-mediatedprocesses. Thus, it is well known in the art that the N-terminalmethionine encoded by the translation initiation codon generally isremoved with high efficiency from any protein after translation in alleukaryotic cells. While the N-terminal methionine on most proteins-alsois efficiently removed in most prokaryotes, for some proteins thisprokaryotic removal process is inefficient, depending on the nature ofthe amino acid to which the N-terminal methionine is covalently linked.

In addition to encompassing host cells containing the vector constructsdiscussed herein, the invention also encompasses primary, secondary, andimmortalized host cells of vertebrate origin, particularly mammalianorigin, that have been engineered to delete or replace endogenousgenetic material, and/or to include genetic material (e.g., heterologouspolynucleotide sequences) that is operably associated withpolynucleotides of the invention, and which activates, alters, and/oramplifies endogenous polynucleotides of the present invention. Forexample, techniques known in the art may be used to operably associateheterologous control regions (e.g., promoter and/or enhancer) andendogenous polynucleotide sequences of the present invention viahomologous recombination, resulting in the formation of a newtranscription unit (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24,1997; U.S. Pat. No. 5,733,761, issued Mar. 31, 1998; InternationalPublication No. WO 96/29411, published Sep. 26, 1996; InternationalPublication No. WO 94/12650, published Aug. 4, 1994; Koller et al.,Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijistra et al.,Nature 342:435-438 (1989), the disclosures of each of which areincorporated by reference in their entireties).

In addition, polypeptides of the invention can be chemically synthesizedusing any effective technique (e.g., see Creighton, 1983, Proteins:Structures and Molecular Principles, W.H. Freeman & Co., NY., andHunkapiller et al., Nature, 310:105-111 (1984)). For example, apolypeptide or a fragment of a polypeptide of the present invention canbe synthesized by use of a peptide synthesizer. Furthermore, if desired,nonclassical amino acids or chemical amino acid analogs can beintroduced as a substitution or addition into the polypeptide sequence.Non-classical amino acids include, but are not limited to, to theD-isomers of the common amino acids, 2,4-diaminobutyric acid,alpha-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyricacid, alpha-Abu, alpha-Ahx, 6-amino hexanoic acid, Aib, 2-aminoisobutyric acid, 3-amino propionic acid, ornithine, norleucine,norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline,cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,cyclohexylalanine, alpha-alanine, fluoro-amino acids, designer aminoacids such as alpha-methyl amino acids, Ca-methyl amino acids, Na-methylamino acids, and amino acid analogs in general. Furthermore, the aminoacid can be D (dextrorotary) or L (levorotary).

Non-naturally occurring variants may be produced using art-knownmutagenesis techniques, which include, but are not limited tooligonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis,site directed mutagenesis (see, e.g., Carter et al., Nucl. Acids Res.13:4331 (1986); and Zoller et al., Nucl. Acids Res. 10:6487 (1982)),cassette mutagenesis (see, e.g., Wells et al., Gene 34:315 (1985)),restriction selection mutagenesis (see, e.g., Wells et al., Philos.Trans. R. Soc. London SerA 317:415 (1986)).

The invention encompasses polypeptides which are differentially modifiedduring or after translation, e.g., by glycosylation, acetylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to an antibody molecule or othercellular ligand, etc. Any of numerous chemical modifications may becarried out by known techniques, including but not limited, to specificchemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8protease, NaBH4; acetylation, formylation, oxidation, reduction;metabolic synthesis in the presence of tunicamycin; etc.

Additional post-translational modifications encompassed by the inventioninclude, for example, e.g., N-linked or O-linked carbohydrate chains,processing of N-terminal or C-terminal ends), attachment of chemicalmoieties to the amino acid backbone, chemical modifications of N-linkedor O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of procaryotic host cellexpression. The polypeptides may also be modified with a detectablelabel, such as an enzymatic, fluorescent, isotopic or affinity label toallow for detection and isolation of the protein.

Also provided by the invention are chemically modified derivatives ofthe polypeptides of the invention which may provide additionaladvantages such as increased solubility, stability and circulating timeof the polypeptide, or decreased immunogenicity (see U.S. Pat. No.4,179,337). The chemical moieties for derivitization may be selectedfrom water soluble polymers such as polyethylene glycol, ethyleneglycol/propylene glycol copolymers, carboxymethylcellulose, dextran,polyvinyl alcohol and the like. The polypeptides may be modified atrandom positions within the molecule, or at predetermined positionswithin the molecule and may include one, two, three or more attachedchemical moieties.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 1 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog). For example,the polyethylene glycol may have an average molecular weight of about200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000,11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500,16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000,25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000,75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.

As noted above, the polyethylene glycol may have a branched structure.Branched polyethylene glycols are described, for example, in U.S. Pat.No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72(1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750 (1999);and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999), the disclosuresof each of which are incorporated herein by reference.

The polyethylene glycol molecules (or other chemical moieties) should beattached to the protein with consideration of effects on functional orantigenic domains of the protein. There are a number of attachmentmethods available to those skilled in the art, e.g., EP 0 401 384,herein incorporated by reference (coupling PEG to G-CSF), see also Maliket al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation ofGM-CSF using tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group may include lysine residues and theN-terminal amino acid residues; those having a free carboxyl group mayinclude aspartic acid residues glutamic acid residues and the C-terminalamino acid residue. Sulfhydryl groups may also be used as a reactivegroup for attaching the polyethylene glycol molecules. Preferred fortherapeutic purposes is attachment at an amino group, such as attachmentat the N-terminus or lysine group.

As suggested above, polyethylene glycol may be attached to proteins vialinkage to any of a number of amino acid residues. For example,polyethylene glycol can be linked to a proteins via covalent bonds tolysine, histidine, aspartic acid, glutamic acid, or cysteine residues.One or more reaction chemistries may be employed to attach polyethyleneglycol to specific amino acid residues (e.g., lysine, histidine,aspartic acid, glutamic acid, or cysteine) of the protein or to morethan one type of amino acid residue (e.g., lysine, histidine, asparticacid, glutamic acid, cysteine and combinations thereof) of the protein.

One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration of the presentcomposition, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (polypeptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective proteins chemicallymodified at the N-terminus modification may be accomplished by reductivealkylation which exploits differential reactivity of different types ofprimary amino groups (lysine versus the N-terminal) available forderivatization in a particular protein. Under the appropriate reactionconditions, substantially selective derivatization of the protein at theN-terminus with a carbonyl group containing polymer is achieved.

As indicated above, pegylation of the proteins of the invention may beaccomplished by any number of means. For example, polyethylene glycolmay be attached to the protein either directly or by an interveninglinker. Linkerless systems for attaching polyethylene glycol to proteinsare described in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys.9:249-304 (1992); Francis et al., Intern. J. of Hematol. 68:1-18 (1998);U.S. Pat. No. 4,002,531; U.S. Pat. No. 5,349,052; WO 95/06058; and WO98/32466, the disclosures of each of which are incorporated herein byreference.

One system for attaching polyethylene glycol directly to amino acidresidues of proteins without an intervening linker employs tresylatedMPEG, which is produced by the modification of monmethoxy polyethyleneglycol (MPEG) using tresylchloride (CISO.sub.2 CH.sub.2 CF.sub.3). Uponreaction of protein with tresylated MPEG, polyethylene glycol isdirectly attached to amine groups of the protein. Thus, the inventionincludes protein-polyethylene glycol conjugates produced by reactingproteins of the invention with a polyethylene glycol molecule having a2,2,2-trifluoreothane sulphonyl group.

Polyethylene glycol can also be attached to proteins using a number ofdifferent intervening linkers. For example, U.S. Pat. No. 5,612,460, theentire disclosure of which is incorporated herein by reference,discloses urethane linkers for connecting polyethylene glycol toproteins. Protein-polyethylene glycol conjugates wherein thepolyethylene glycol is attached to the protein by a linker can also beproduced by reaction of proteins with compounds such asMPEG-succinimidylsuccinate, MPEG activated with1,1′-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate,MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives. Anumber additional polyethylene glycol derivatives and reactionchemistries for attaching polyethylene glycol to proteins are describedin WO 98/32466, the entire disclosure of which is incorporated herein byreference. Pegylated protein products produced using the reactionchemistries set out herein are included within the scope of theinvention.

The number of polyethylene glycol moieties attached to each protein ofthe invention (i.e., the degree of substitution) may also vary. Forexample, the pegylated proteins of the invention may be linked, onaverage, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or morepolyethylene glycol molecules. Similarly, the average degree ofsubstitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9,8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or18-20 polyethylene glycol moieties per protein molecule. Methods fordetermining the degree of substitution are discussed, for example, inDelgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).

The polypeptides of the invention may be in monomers or multimers (i.e.,dimers, trimers, tetramers and higher multimers). Accordingly, thepresent invention relates to monomers and multimers of the polypeptidesof the invention, their preparation, and compositions containing them.In specific embodiments, the polypeptides of the invention are monomers,dimers, trimers or tetramers. In additional embodiments, the multimersof the invention are at least dimers, at least trimers, or at leasttetramers.

Multimers encompassed by the invention may be homomers or heteromers. Asused herein, the term homomer, refers to a multimer containing onlypolypeptides having an identical sequence.

As used herein, the term heteromer refers to a multimer containing oneor more heterologous polypeptides (i.e., polypeptides having differentsequences). In a specific embodiment, the multimer of the invention is aheterodimer, a heterotrimer, or a heterotetramer. In additionalembodiments, the heteromeric multimer of the invention is at least aheterodimer, at least a heterotrimer, or at least a heterotetramer.

Any effective multimer may be formed. For example, in one embodiment ofthe present invention, IFN-λs form multimers of and between two of thethree members, including, for example, IFN-λ1/IFN-λ2, IFN-λ1/IFN-λ3 andIFN-λ2/IFN-λ3, or multimers with all three members of the IFN-λ family.In another embodiment of the present invention an effective multimercontains other members of the cytokine family, including the IFN andIL-10 families of cytokines.

Multimers of the invention may be the result of hydrophobic,hydrophilic, ionic and/or covalent associations and/or may be indirectlylinked, by for example, liposome formation. Thus, in one embodiment,multimers of the invention, such as, for example, homodimers orhomotrimers, are formed when polypeptides of the invention contact oneanother in solution. In another embodiment, heteromultimers of theinvention, such as, for example, heterotrimers or heterotetramers, areformed when polypeptides of the invention contact antibodies to thepolypeptides of the invention (including antibodies to the heterologouspolypeptide sequence in a fusion protein of the invention) in solution.In other embodiments, multimers of the invention are formed by covalentassociations with and/or between the polypeptides of the invention. Suchcovalent associations may involve one or more amino acid residuescontained in the polypeptide sequence. In one instance, the covalentassociations are cross-linking between cysteine residues located withinthe polypeptide sequences which interact in the native (i.e., naturallyoccurring) polypeptide. In another instance, the covalent associationsare the consequence of chemical or recombinant manipulation.Alternatively, such covalent associations may involve one or more aminoacid residues contained in the heterologous polypeptide sequence in afusion protein of the present invention. In one example, covalentassociations are between the heterologous sequence contained in a fusionprotein of the invention (see, e.g., U.S. Pat. No. 5,478,925). In aspecific example, the covalent associations are between the heterologoussequence contained in an −Fc fusion protein of the invention (asdescribed herein). In another specific example. covalent associations offusion proteins of the invention are between heterologous polypeptidesequences from another protein that is capable of forming covalentlyassociated multimers, such as for example, oseteoprotegerin (see, e.g.,International Publication NO: WO 98/49305, the contents of which areherein incorporated by reference in its entirety). In anotherembodiment, two or more polypeptides of the invention are joined throughpeptide linkers. Examples include those peptide linkers described inU.S. Pat. No. 5,073,627 (hereby incorporated by reference). Proteinscomprising multiple polypeptides of the invention separated by peptidelinkers may be produced using conventional recombinant DNA technology.

In another example, proteins of the invention are associated byinteractions between FLAG polypeptide sequence contained in fusionproteins of the invention containing FLAG polypeptide sequence. In afurther embodiment, associations proteins of the invention areassociated by interactions between heterologous polypeptide sequencecontained in FLAG fusion proteins of the invention and anti-FLAGantibody.

Further methods that may be used for production multimers of the presentinvention are disclosed in U.S. Pat. No. 6,433,145, and are herebyincorporated by reference in their entirety.

Antibodies

The polypeptides of the invention also relate to antibodies and T-cellantigen receptors (TCR) which immunospecifically bind a polypeptide,polypeptide fragment and/or an epitope of a polypeptide of the presentinvention (as determined by immunoassays well known in the art forassaying specific antibody-antigen binding).

The term “antibody,” as used herein, refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that immunospecificallybinds an antigen. The immunoglobulin molecules of the invention can beof any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1,IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.In a specific embodiment, the immunoglobulin molecules of the inventionare IgG1. In another specific embodiment, the immunoglobulin moleculesof the invention are IgG4.

Antibodies of the invention include, but are not limited to, polyclonal,monoclonal, multispecific, human, humanized or chimeric antibodies,single chain antibodies, Fab fragments, F(ab) fragments, fragmentsproduced by a Fab expression library, anti-idiotypic (anti-Id)antibodies (including, e.g., anti-Id antibodies to antibodies of theinvention), and epitope-binding fragments of any of the above.

Most preferably the antibodies are human antigen-binding antibodyfragments of the present invention and include, but are not limited to,Fab, Fab′ and F(ab)₂, Fd, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv) and fragments comprising eithera VL or VH domain. Antigen-binding antibody fragments, includingsingle-chain antibodies, may comprise the variable region(s) alone or incombination with the entirety or a portion of the following: hingeregion, CH1, CH2, and CH3 domains. Also included in the invention areantigen-binding fragments also comprising any combination of variableregion(s) with a hinge region, CH1, CH21 and CH3 domains. The antibodiesof the invention may be from any animal origin including birds andmammals. Preferably, the antibodies are human, murine (e.g., mouse andrat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken.As used herein, “human” antibodies include antibodies having the aminoacid sequence of a human immunoglobulin and include antibodies isolatedfrom human immunoglobulin libraries or from animals transgenic for oneor more human immunoglobulin and that do not express endogenousimmunoglobulins, as described infra and, for example in, U.S. Pat. No.5,939,598 by Kucherlapati et al.

The antibodies of the present invention may be monospecific, bispecific,trispecific or of greater multispecificity. Multispecific antibodies maybe specific for different epitopes of a polypeptide of the presentinvention or may be specific for both a polypeptide of the presentinvention as well as for a heterologous epitope, such as a heterologouspolypeptide or solid support material. See, e.g., PCT publications WO93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J.Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681;4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol.148:1547-1553 (1992).

Antibodies of the present invention may be described or specified interms of the epitope(s) or portion(s) of a polypeptide of the presentinvention that they recognize or specifically bind. The epitope(s) orpolypeptide portion(s) may be specified as described herein, e.g., byN-terminal and C-terminal positions, by size in contiguous amino acidresidues. Preferred epitopes of the invention include those that includea sequence homology between IFN-λ forms and alleles. Also preferred areepitopes that include sequence differences between IFN-λ forms andalleles. These can be ascertained by those of ordinary skill in the art,and are further described in the polypeptide section of the presentspecification.

Antibodies of the present invention may also be described or specifiedin terms of their cross-reactivity. Antibodies that do not bind anyother analog, ortholog, or homolog of a polypeptide of the presentinvention are included. Antibodies that bind polypeptides with at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 65%, at least 60%, at least 55%, and at least 50% identity(as calculated using methods known in the art and described herein) to apolypeptide of the present invention are also included in the presentinvention. In specific embodiments, antibodies of the present inventioncross-react with murine, rat and/or rabbit homologs of human proteinsand the corresponding epitopes thereof. Antibodies that do not bindpolypeptides with less than 95%, less than 90%, less than 85%, less than80%, less than 75%, less than 70%, less than 65%, less than 60%, lessthan 55%, and less than 50% identity (as calculated using methods knownin the art and described herein) to a polypeptide of the presentinvention are also included in the present invention. In a specificembodiment, the above-described cross-reactivity is with respect to anysingle specific antigenic or immunogenic polypeptide, or combination(s)of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenicpolypeptides disclosed herein. Further included in the present inventionare antibodies which bind polypeptides encoded by polynucleotides whichhybridize to a polynucleotide of the present invention under stringenthybridization conditions (as described herein).

Antibodies of the present invention may also be described or specifiedin terms of their binding affinity to a polypeptide of the invention.Preferred binding affinities include those with a dissociation constantor Kd less than 5×10² M, 10⁻² M, 5×10⁻³ M, 10⁻³M, 5×10⁻⁴ M, 10⁻⁴ M,5×10⁻⁵M, 5×10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M,5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M,10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M.

The invention also provides antibodies that competitively inhibitbinding of an antibody to an epitope of the invention as determined byany method known in the art for determining competitive binding, forexample, the immunoassays described herein, or the ability of theantibody to block the biological activity of the CRF2-12 receptor orreceptor complex, the ability to block binding of the polypeptides ofthe present invention to the CRF2-12 receptor or receptor complex, orthe ability of the antibody to block signal transduction from thereceptor or receptor complex. The antibodies may be directed eithertowards the receptor or the IFN-λ polypeptides of the present invention.In preferred embodiments, the antibody competitively inhibits binding tothe epitope by at least 95%, at least 90%, at least 85%, at least 80%,at least 75%, at least 70%, at least 60%, or at least 50%.

Antibodies of the present invention may act as agonists or antagonistsof the polypeptides of the present invention. For example, the presentinvention includes antibodies that disrupt the receptor/ligandinteractions with the polypeptides of the invention either partially orfully. Preferrably, antibodies of the present invention bind anantigenic epitope disclosed herein, or a portion thereof. The inventionfeatures both receptor-specific antibodies and ligand-specificantibodies. The invention also features receptor-specific antibodiesthat do not prevent ligand binding but prevent receptor activation.Receptor activation (i.e., signaling) may be determined by techniquesdescribed herein or otherwise known in the art. For example, receptoractivation can be determined by detecting the phosphorylation (e.g.,tyrosine or serine/threonine) of the receptor or its substrate byimmunoprecipitation followed by western blot analysis (for example, asdescribed supra, or in the EXAMPLES section infra). In specificembodiments, antibodies are provided that inhibit ligand activity orreceptor activity by at least 95%, at least 90%, at least 85%, at least80%, at least 75%, at least 70%, at least 60%, or at least 50% of theactivity in absence of the antibody.

The invention also features receptor-specific antibodies which bothprevent ligand binding and receptor activation as well as antibodiesthat recognize the receptor-ligand complex, and, preferably, do notspecifically recognize the unbound receptor or the unbound ligand.Likewise, included in the invention are neutralizing antibodies whichbind the ligand and prevent binding of the ligand to the receptor, aswell as antibodies which bind the ligand, thereby preventing receptoractivation, but do not prevent the ligand from binding the receptor.Further included in the invention are antibodies that activate thereceptor. These antibodies may act as receptor agonists, i.e.,potentiate or activate either all or a subset of the biologicalactivities of the ligand-mediated receptor activation, for example, byinducing dimerization of the receptor. The antibodies may be specifiedas agonists, antagonists or inverse agonists for biological activitiescomprising the specific biological activities of the peptides of theinvention disclosed herein. The above antibody agonists can be madeusing methods known in the art. See, e.g., PCT publication WO 96/40281;U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chenet al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol.161(4):1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214(1998); Yoon et al., J. Immunol. 160(7):3170-3179 (1998); Prat et al.,J. Cell. Sci. 111(Pt2):237-247 (1998); Pitard et al., J. Immunol.Methods 205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241(1997); Carlson et al., J. Biol. Chem. 272(17): 11295-11301 (1997);Taryman et al., Neuron 14(4):755-762 (1995); Muller et al., Structure6(9):1153-1167 (1998); Bartunek et al., Cytokine 8(1):14-20 (1996)(which are all incorporated by reference herein in their entireties).

Antibodies of the present invention may be used, for example, but notlimited to, to purify, detect, and target the polypeptides of thepresent invention, including both in vitro and in vivo diagnostic andtherapeutic methods. For example, the antibodies have use inimmunoassays for qualitatively and quantitatively measuring levels ofthe polypeptides of the present invention in biological samples. See,e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988) (incorporated by reference hereinin its entirety).

As discussed in more detail below, the antibodies of the presentinvention may be used either alone or in combination with othercompositions. The antibodies may further be recombinantly fused to aheterologous polypeptide at the N- or C-terminus or chemicallyconjugated (including covalently and non-covalently conjugations) topolypeptides or other compositions. For example, antibodies of thepresent invention may be recombinantly fused or conjugated to moleculesuseful as labels in detection assays and effector molecules such asheterologous polypeptides, drugs, radionuclides, or toxins. See, e.g.,PCT publications WO. 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No.5,314,995; and EP 396,387.

The antibodies of the invention include derivatives that are modified,i.e., by the covalent attachment of any type of molecule to the antibodysuch that covalent attachment does not prevent the antibody fromgenerating an anti-idiotypic response. For example, but not by way oflimitation, the antibody derivatives include antibodies that have beenmodified, e.g., by glycosylation, acetylation, pegylation,phosphylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

The antibodies of the present invention may be generated by any suitablemethod known in the art. Polyclonal antibodies to an antigen-of-interestcan be produced by various procedures well known in the art. Forexample, a polypeptide of the invention can be administered to varioushost animals including, but not limited to, rabbits, mice, rats, etc. toinduce the production of sera containing polyclonal antibodies specificfor the antigen. Various adjuvants may be used to increase theimmunological response, depending on the host species, and include butare not limited to, Freund's (complete and incomplete), mineral gelssuch as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and corynebacteriumparvum. Such adjuvants are also well known in the art.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling, et al., in: Monoclonal Antibodies and T-CellHybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporatedby reference in their entireties). The term “monoclonal antibody” asused herein is not limited to antibodies produced through hybridomatechnology. The term “monoclonal antibody” refers to an antibody that isderived from a single clone, including any eukaryotic, prokaryotic, orphage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art and arediscussed in detail in the Examples. In a non-limiting example, mice canbe immunized with a polypeptide of the invention or a cell expressingsuch peptide. Once an immune response is detected, e.g., antibodiesspecific for the antigen are detected in the mouse serum, the mousespleen is harvested and splenocytes isolated. The splenocytes are thenfused by well known techniques to any suitable myeloma cells, forexample cells from cell line SP20 available from the ATCC. Hybridomasare selected and cloned by limited dilution. The hybridoma clones arethen assayed by methods known in the art for cells that secreteantibodies capable of binding a polypeptide of the invention. Ascitesfluid, which generally contains high levels of antibodies, can begenerated by immunizing mice with positive hybridoma clones.

Accordingly, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody of theinvention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with an antigen of theinvention with myeloma cells and then screening the hybridomas resultingfrom the fusion for hybridoma clones that secrete an antibody able tobind a polypeptide of the invention. Antibody fragments which recognizespecific epitopes may be generated by known techniques. For example, Faband F(ab′)2 fragments of the invention may be produced by proteolyticcleavage of immunoglobulin molecules, using enzymes such as papain (toproduce Fab fragments) or pepsin (to produce F(ab′)2 fragments). F(ab′)2fragments contain the variable region, the light chain constant regionand the CH1 domain of the heavy chain.

For example, the antibodies of the present invention can also begenerated using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of phage particles that carry the polynucleotide sequencesencoding them. In a particular embodiment, such phage can be utilized todisplay antigen binding domains expressed from a repertoire orcombinatorial antibody library (e.g., human or murine). Phage expressingan antigen binding domain that binds the antigen of interest can beselected or identified with antigen, e.g., using labeled antigen orantigen bound or captured to a solid surface or bead. Phage used inthese methods are typically filamentous phage including fd and M13binding domains expressed from phage with Fab, Fv or disulfidestabilized Fv antibody domains recombinantly fused to either the phagegene III or gene VIII protein. Examples of phage display methods thatcan be used to make the antibodies of the present invention includethose disclosed in Brinkman et al., J. Immunol. Methods 182:41-50(1995); Ames et al., J. Immunol. Methods 184:177-186 (1995);Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al.,Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280(1994); PCT application No. PCT/GB91/01134; PCT publications WO90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108;each of which is incorporated herein by reference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab)₂ fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869(1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al.,Science 240:1041-1043 (1988) (said references incorporated by referencein their entireties).

Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu etal., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040(1988). For some uses, including in vivo use of antibodies in humans andin vitro detection assays, it may be preferable to use chimeric,humanized, or human antibodies. A chimeric antibody is a molecule inwhich different portions of the antibody are derived from differentanimal species, such as antibodies having a variable region derived froma murine monoclonal antibody and a human immunoglobulin constant region.Methods for producing chimeric antibodies are known in the art. Seee.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214(1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S.Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporatedherein by reference in their entirety. Humanized antibodies are antibodymolecules from non-human species that bind the desired antigen havingone or more complementarity determining regions (CDRs) from thenon-human species and framework regions from a human immunoglobulinmolecule. Often, framework residues in the human framework regions willbe substituted with the corresponding residue from the CDR donorantibody to alter, preferably improve, antigen binding. These frameworksubstitutions are identified by methods well known in the art, e.g., bymodeling of the interactions of the CDR and framework residues toidentify framework residues important for antigen binding and sequencecomparison to identify unusual framework residues at particularpositions; (See, e.g., Queen et al. U.S. Pat. No. 5,585,089; Riechmannet al., Nature 332:323 (1988), which are incorporated herein byreference in their entireties.) Antibodies can be humanized using avariety of techniques known in the art including, for example,CDR-grafting (EP 239,400; International Publication No. WO91/09967; U.S.Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing(EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,332).

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedabove using antibody libraries derived from human immunoglobulinsequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCTpublications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO96/34096, WO 96/33735, and WO 91/10741; each of which is incorporatedherein by reference in its entirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring which express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained from the immunized,transgenic mice using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923;5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318;5,885,793; 5,916,771; and 5,939,598, which are incorporated by referenceherein in their entirety. In addition, companies such as Abgenix. Inc.(Freemont, Calif.) and Genpharm (San Jose, Calif.) can be engaged toprovide human antibodies directed against a selected antigen usingtechnology similar to that described above.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/technology 12:899-903(1988)).

Further, antibodies to the polypeptides of the invention can, in turn,be utilized to generate anti-idiotype antibodies that “mimic”polypeptides of the invention using techniques well known to thoseskilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444;(1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example,antibodies which bind to and competitively inhibit polypeptidemultimerization and/or binding of a polypeptide of the invention to aligand can be used to generate anti-idiotypes that “mimic” thepolypeptide multimerization and/or binding domain and, as a consequence,bind to and neutralize polypeptide and/or its ligand. Such neutralizinganti-idiotypes or Fab fragments of such anti-idiotypes can be used intherapeutic regimens to neutralize polypeptide ligand. For example, suchanti-idiotypic antibodies can be used to bind a polypeptide of theinvention and/or to bind its ligands/receptors, and thereby block itsbiological activity.

Polynucleotides Encoding Antibodies

The invention further provides polynucleotides comprising a nucleotidesequence encoding an antibody of the invention and fragments thereof.The invention also encompasses polynucleotides that hybridize understringent or lower stringency hybridization conditions, e.g., as definedsupra, to polynucleotides that encode an antibody, preferably, thatspecifically binds to a polypeptide of the invention.

The polynucleotides may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. For example,if the nucleotide sequence of the antibody is known, a polynucleotideencoding the antibody may be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier et al., BioTechniques17:242 (1994)), which, briefly, involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligating of those oligonucleotides, and thenamplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody may be generatedfrom nucleic acid from a suitable source. If a clone containing anucleic acid encoding a particular antibody is not available, but thesequence of the antibody molecule is known, a nucleic acid encoding theimmunoglobulin may be chemically synthesized or obtained from a suitablesource (e.g., an antibody cDNA library, or a cDNA library generatedfrom, or nucleic acid, preferably poly A+RNA, isolated from, any tissueor cells expressing the antibody, such as hybridoma cells selected toexpress an antibody of the invention) by PCR amplification usingsynthetic primers hybridizable to the 3′ and 5′ ends of the sequence orby cloning using an oligonucleotide probe specific for the particulargene sequence to identify, e.g., a cDNA clone from a cDNA library thatencodes the antibody. Amplified nucleic acids generated by PCR may thenbe cloned into replicable cloning vectors using any method well known inthe art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe antibody is determined, the nucleotide sequence of the antibody maybe manipulated using methods well known in the art for the manipulationof nucleotide sequences, e.g., recombinant DNA techniques, site directedmutagenesis, PCR, etc. (see, for example, the techniques described inSambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel etal., eds., 1998, Current Protocols in Molecular Biology. John Wiley &Soils, NY, which are both incorporated by reference herein in theirentireties), to generate antibodies having a different amino acidsequence, for example to create amino acid substitutions, deletions,and/or insertions.

In a specific embodiment, the amino acid sequence of the heavy and/orlight chain variable domains may be inspected to identify the sequencesof the complementarity determining regions (CDRs) by methods that arewell know in the art, e.g., by comparison to known amino acid sequencesof other heavy and light chain variable regions to determine the regionsof sequence hypervariability. Using routine recombinant DNA techniques,one or more of the CDRs may be inserted within framework regions, e.g.,into human framework regions to humanize a non-human antibody, asdescribed supra. The framework regions may be naturally occurring orconsensus framework regions, and preferably human framework regions(see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for alisting of human framework regions). Preferably, the polynucleotidegenerated by the combination of the framework regions and CDRs encodesan antibody that specifically binds a polypeptide of the invention.Preferably, as discussed supra, one or more amino acid substitutions maybe made within the framework regions, and, preferably, the amino acidsubstitutions improve binding of the antibody to its antigen.Additionally, such methods may be used to make amino acid substitutionsor deletions of one or more variable region cysteine residuesparticipating in an intrachain disulfide bond to generate antibodymolecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentinvention and within the skill of the art.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984);Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature314:452-454 (1985)) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Asdescribed supra, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine mAb and a human immunoglobulinconstant region, e.g., humanized antibodies.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-42 (1988);Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Wardet al., Nature 334:544-54 (1989)) can be adapted to produce single chainantibodies. Single chain antibodies are formed by linking the heavy andlight chain fragments of the Fv region via an amino acid bridge,resulting in a single chain polypeptide. Techniques for the assembly offunctional Fv fragments in E. coli may also be used (Skerra et al.,Science 242:1038-1041 (1988)).

Assays for Antibody Binding

The antibodies of the invention may be assayed for immunospecificbinding by any method known in the art. The immunoassays which can beused include but are not limited to competitive and non-competitiveassay systems using techniques such as western blots, radioimmunoassays,ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Ausubel et al, eds, 1994,Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,New York, which is incorporated by reference herein in its entirety).Exemplary immunoassays are described briefly below (but are not intendedby way of limitation).

Immunoprecipitation protocols generally comprise lysing a population ofcells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100,1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphateat pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/orprotease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate),adding the antibody of interest to the cell lysate, incubating for aperiod of time (e.g., 14 hours) at 4.degree. C., adding protein A and/orprotein G sepharose beads to the cell lysate, incubating for about anhour or more at 4.degree. C., washing the beads in lysis buffer andresuspending the beads in SDS/sample buffer. The ability of the antibodyof interest to immunoprecipitate a particular antigen can be assessedby, e.g., western blot analysis. One of skill in the art would beknowledgeable as to the parameters that can be modified to increase thebinding of the antibody to an antigen and decrease the background (e.g.,pre-clearing the cell lysate with sepharose beads). For furtherdiscussion regarding immunoprecipitation protocols see, e.g., Ausubel etal, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.16.1.

Western blot analysis generally comprises preparing protein samples,electrophoresis of the protein samples in a polyacrylamide gel (e.g.,8%-20% SDS-PAGE depending on the molecular weight of the antigen),transferring the protein sample from the polyacrylamide gel to amembrane such as nitrocellulose, PVDF or nylon, blocking the membrane inblocking solution (e.g., PBS with 3% BSA or non-fat milk), washing themembrane in washing buffer (e.g., PBS-Tween 20), blocking the membranewith primary antibody (the antibody of interest) diluted in blockingbuffer, washing the membrane in washing buffer, blocking the membranewith a secondary antibody (which recognizes the primary antibody, e.g.,an anti-human antibody) conjugated to an enzymatic substrate (e.g.,horseradish peroxidase or alkaline phosphatase) or radioactive molecule(e.g., 32P or 125I) diluted in blocking buffer, washing the membrane inwash buffer, and detecting the presence of the antigen. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected and to reduce the background noise. Forfurther discussion regarding western blot protocols see, e.g., Ausubelet al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.8.1.

ELISAs comprise preparing antigen, coating a surface, for example thewell of a 96 well microtiter plate, with the antigen, adding theantibody of interest conjugated to a detectable compound such as anenzymatic substrate (e.g., horseradish peroxidase or alkalinephosphatase) to the well and incubating for a period of time, anddetecting the presence of the antigen. In ELISAs the antibody ofinterest does not have to be conjugated to a detectable compound;instead, a second antibody (which recognizes the antibody of interest)conjugated to a detectable compound may be added to the well. Further,instead of coating the well with the antigen, the antibody may be coatedto the well. In this case, a second antibody conjugated to a detectablecompound may be added following the addition of the antigen of interestto the coated well. One of skill in the art would be knowledgeable as tothe parameters that can be modified to increase the signal detected aswell as other variations of ELISAs known in the art. For furtherdiscussion regarding ELISAs see, e.g., Ausubel et al, eds, 1994, CurrentProtocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., NewYork at 11.2.1.

The binding affinity of an antibody to an antigen and the off-rate of anantibody-antigen interaction can be determined by competitive bindingassays. One example of a competitive binding assay is a radioimmunoassaycomprising the incubation of labeled antigen (e.g., 3H or 125I) with theantibody of interest in the presence of increasing amounts of unlabeledantigen, and the detection of the antibody bound to the labeled antigen.The affinity of the antibody of interest for a particular antigen andthe binding off-rates can be determined from the data by scatchard plotanalysis. Competition with a second antibody can also be determinedusing radioimmunoassays. In this case, the antigen is incubated withantibody of interest conjugated to a labeled compound (e.g., 3H or 125I)in the presence of increasing amounts of an unlabeled second antibody.

Diagnosis and Imaging

Labeled antibodies, and derivatives and analogs thereof, whichspecifically bind to a polypeptide of interest can be used fordiagnostic purposes to detect, diagnose, or monitor diseases, disorders,and/or conditions associated with the aberrant expression and/oractivity of a polypeptide of the invention. The invention provides forthe detection of aberrant expression of a polypeptide of interest,comprising (a) assaying the expression of the polypeptide of interest incells or body fluid of an individual using one or more antibodiesspecific to the polypeptide interest and (b) comparing the level of geneexpression with a standard gene expression level, whereby an increase ordecrease in the assayed polypeptide gene expression level compared tothe standard expression level is indicative of aberrant expression.

The invention provides a diagnostic assay for diagnosing a disorder,comprising (a) assaying the expression of the polypeptide of interest incells or body fluid of an individual using one or more antibodiesspecific to the polypeptide interest and (b) comparing the level of geneexpression with a standard gene expression level, whereby an increase ordecrease in the assayed polypeptide gene expression level compared tothe standard expression level is indicative of a particular disorder.With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Antibodies of the invention can be used to assay protein levels in abiological sample using classical immunohistological methods known tothose of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, et al., J. Cell. Biol. 105:3087-3096(1987)). Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase; radioisotopes, such as iodine (125I, 121I), carbon (14C),sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc);luminescent labels, such as luminol; and fluorescent labels, such asfluorescein and rhodamine, and biotin.

One aspect of the invention is the detection and diagnosis of a diseaseor disorder associated with aberrant expression of a polypeptide ofinterest in an animal, preferably a mammal and most preferably a human.In one embodiment, diagnosis comprises: a) administering (for example,parenterally, subcutaneously, or intraperitoneally) to a subject aneffective amount of a labeled molecule which specifically binds to thepolypeptide of interest; b) waiting for a time interval following theadministering for permitting the labeled molecule to preferentiallyconcentrate at sites in the subject where the polypeptide is expressed(and for unbound labeled molecule to be cleared to background level); c)determining background level; and d) detecting the labeled molecule inthe subject, such that detection of labeled molecule above thebackground level indicates that the subject has a particular disease ordisorder associated with aberrant expression of the polypeptide ofinterest. Background level can be determined by various methodsincluding, comparing the amount of labeled molecule detected to astandard value previously determined for a particular system.

It will be understood in the art that the size of the subject and theimaging system used will determine the quantity of imaging moiety neededto produce diagnostic images. In the case of a radioisotope moiety, fora human subject, the quantity of radioactivity injected will normallyrange from about 5 to 20 millicuries of 99 mTc. The labeled antibody orantibody fragment will then preferentially accumulate at the location ofcells which contain the specific protein. In vivo tumor imaging isdescribed in S. W. Burchiel et al. “Immunopharmacokinetics ofRadiolabeled Antibodies and Their Fragments.” (Chapter 13 in TumorImaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A.Rhodes, eds., Masson Publishing Inc. (1982).

Depending on several variables, including the type of label used and themode of administration, the time interval following the administrationfor permitting the labeled molecule to preferentially concentrate atsites in the subject and for unbound labeled molecule to be cleared tobackground level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. Inanother embodiment the time interval following administration is 5 to 20days or 5 to 10 days.

In an embodiment, monitoring of the disease or disorder is carried outby repeating the method for diagnosing the disease or disease, forexample, one month after initial diagnosis, six months after initialdiagnosis, one year after initial diagnosis, etc.

Presence of the labeled molecule can be detected in the patient usingmethods known in the art for in vivo scanning. These methods depend uponthe type of label used. Skilled artisans will be able to determine theappropriate method for detecting a particular label. Methods and devicesthat may be used in the diagnostic methods of the invention include, butare not limited to, computed tomography (CT), whole body scan such asposition emission tomography (PET), magnetic resonance imaging (MRI),and sonography.

In a specific embodiment, the molecule is labeled with a radioisotopeand is detected in the patient using a radiation responsive surgicalinstrument (Thurston et al., U.S. Pat. No. 5,441,050). In anotherembodiment, the molecule is labeled with a fluorescent compound and isdetected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patent using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

Kits

The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises an antibody of theinvention, preferably a purified antibody, in one or more containers. Ina specific embodiment, the kits of the present invention contain asubstantially isolated polypeptide comprising an epitope which isspecifically immunoreactive with an antibody included in the kit.Preferably, the kits of the present invention further comprise a controlantibody which does not react with the polypeptide of interest. Inanother specific embodiment, the kits of the present invention contain ameans for detecting the binding of an antibody to a polypeptide ofinterest (e.g., the antibody may be conjugated to a detectable substratesuch as a fluorescent compound, an enzymatic substrate, a radioactivecompound or a luminescent compound, or a second antibody whichrecognizes the first antibody may be conjugated to a detectablesubstrate).

In another embodiment, a kit comprises a set of antibodies that canspecifically bind, and thus distinguish between, the IFN-λ1, IFN-λ2and/or the IFN-λ3 polypeptides of the present invention. In a specificembodiment, the kits of the present invention contain a substantiallyisolated polypeptide comprising an epitope which is specificallyimmunoreactive with each antibody included in the kit. Preferably, thekits of the present invention further comprise a control antibody whichdoes not react with the polypeptides of interest. In another specificembodiment, the kits of the present invention contain a means fordetecting the binding of an antibody to a polypeptide of interest (e.g.,the antibody may be conjugated to a detectable substrate such as afluorescent compound, an enzymatic substrate, a radioactive compound ora luminescent compound, or a second antibody which recognizes the firstantibody may be conjugated to a detectable substrate).

In another embodiment, a kit comprises a set of antibodies that canspecifically bind, and thus distinguish between, alleles of IFN-λ1,IFN-λ2 and/or the IFN-λ3 polypeptides of the present invention. In aspecific embodiment, the kits of the present invention contain asubstantially isolated polypeptide comprising an epitope which isspecifically immunoreactive with each antibody included in the kit.Preferably, the kits of the present invention further comprise a controlantibody which does not react with the polypeptides of interest. Inanother specific embodiment, the kits of the present invention contain ameans for detecting the binding of an antibody to a polypeptide ofinterest (e.g., the antibody may be conjugated to a detectable substratesuch as a fluorescent compound, an enzymatic substrate, a radioactivecompound or a luminescent compound, or a second antibody whichrecognizes the first antibody may be conjugated to a detectablesubstrate).

In another specific embodiment of the present invention, the kit is adiagnostic kit for use in screening serum containing antibodies specificagainst proliferative and/or cancerous polynucleotides and polypeptides.Such a kit may include a control antibody that does not react with thepolypeptide of interest. Such a kit may include a substantially isolatedpolypeptide antigen comprising an epitope which is specificallyimmunoreactive with at least one anti-polypeptide antigen antibody.Further, such a kit includes means for detecting the binding of saidantibody to the antigen (e.g., the antibody may be conjugated to afluorescent compound such as fluorescein or rhodamine which can bedetected by flow cytometry). In specific embodiments, the kit mayinclude a recombinantly produced or chemically synthesized polypeptideantigen. The polypeptide antigen of the kit may also be attached to asolid support.

In a more specific embodiment the detecting means of the above-describedkit includes a solid support to which said polypeptide antigen isattached. Such a kit may also include a non-attached reporter-labeledanti-human antibody. In this embodiment, binding of the antibody to thepolypeptide antigen can be detected by binding of the saidreporter-labeled antibody.

In an additional embodiment, the invention includes a diagnostic kit foruse in screening serum containing antigens of the polypeptide of theinvention. The diagnostic kit includes a substantially isolated antibodyspecifically immunoreactive with polypeptide or polynucleotide antigens,and means for detecting the binding of the polynucleotide or polypeptideantigen to the antibody. In one embodiment, the antibody is attached toa solid support. In a specific embodiment, the antibody may be amonoclonal antibody. The detecting means of the kit may include asecond, labeled monoclonal antibody. Alternatively, or in addition, thedetecting means may include a labeled, competing antigen.

In one diagnostic configuration, test serum is reacted with a solidphase reagent having a surface-bound antigen obtained by the methods ofthe present invention. After binding with specific antigen antibody tothe reagent and removing unbound serum components by washing, thereagent is reacted with reporter-labeled anti-human antibody to bindreporter to the reagent in proportion to the amount of boundanti-antigen antibody on the solid support. The reagent is again washedto remove unbound labeled antibody, and the amount of reporterassociated with the reagent is determined. Typically, the reporter is anenzyme which is detected by incubating the solid phase in the presenceof a suitable fluorometric, luminescent or colorimetric substrate(Sigma, St. Louis, Mo.).

The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, 96-well plate or filter material.These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment of the protein,typically through a free amine group, to a chemically reactive group onthe solid support, such as an activated carboxyl, hydroxyl, or aldehydegroup. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated antigen(s).

Thus, the invention provides an assay system or kit for carrying outthis diagnostic method. The kit generally includes a support withsurface-bound recombinant antigens, and a reporter-labeled anti-humanantibody for detecting surface-bound anti-antigen antibody.

Fusion Proteins

Any polypeptide of the present invention can be used to generate fusionproteins. For example, the polypeptides of the present invention, whenfused to a second protein, can be used as an antigenic tag. Antibodiesraised against the polypeptides of the present invention can be used toindirectly detect the second protein by binding to the polypeptide.Moreover, because secreted proteins target cellular locations based ontrafficking signals, the polypeptides can be used as targeting moleculesonce fused to other proteins.

Examples of domains that can be fused to the polypeptides of the presentinvention include not only heterologous signal sequences, but also otherheterologous functional regions. The fusion does not necessarily need tobe direct, but may occur through linker sequences.

Moreover, fusion proteins may also be engineered to improvecharacteristics of the polypeptides of the present invention. Forinstance, a region of additional amino acids, particularly charged aminoacids, may be added to the N-terminus of the polypeptide to improvestability and persistence during purification from the host cell orsubsequent handling and storage. Also, peptide moieties may be added tothe polypeptide to facilitate purification. Such regions may be removedprior to final preparation of the polypeptide. The addition of peptidemoieties to facilitate handling of polypeptides are familiar and routinetechniques in the art.

The polypeptides of the present invention may be fused with heterologouspolypeptide sequences. For example, the polypeptides of the presentinvention may be fused parts of the constant domain of immunoglobulins(IgA, IgE, IgG, IgM) or portions thereof (CH1, CH2, CH3, and anycombination thereof, including both entire domains and portionsthereof), or albumin (including but not limited to recombinant albumin),resulting in chimeric polypeptides. These fusion proteins facilitatepurification and show an increased half-life in vivo. This has beenshown, e.g., for chimeric proteins consisting of the first two domainsof the human CD4-polypeptide and various domains of the constant regionsof the heavy or light chains of mammalian immunoglobulins (EP A 394,827;Traunecker et al., Nature 331:84-86 (1988)). Fusion proteins that have adisulfide-linked dimeric structure due to the IgG part can also be moreefficient in binding and neutralizing other molecules than the monomericpolypeptide or polypeptide fragment of the present invention alone(Fountoulakis et al., J. Biochem. 270:3958-3964 (1995)). Polynucleotidescomprising or alternatively consisting of nucleic acids which encodethese fusion proteins are also encompassed by the invention

Similarly, EP-A-O 464 533 (Canadian counterpart 2045869) disclosesfusion proteins comprising various portions of constant region ofimmunoglobulin molecules together with another human protein or partthereof. In many cases, the Fc part in a fusion protein is beneficial intherapy and diagnosis, and thus can result in, for example, improvedpharmacokinetic properties. (EP-A 0232 262.) Alternatively, deleting theFc part after the fusion protein has been expressed, detected, andpurified, would be desired. For example, the Fc portion may hindertherapy and diagnosis if the fusion protein is used as an antigen forimmunizations. In drug discovery, for example, human proteins, such ashIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. (See,D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johansonet al., J. Biol. Chem. 270:9459-9471 (1995).)

Moreover, the polypeptides of the present invention can be fused tomarker sequences, such as a peptide which facilitates purification. Inpreferred embodiments, the marker amino acid sequence is ahexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. Another peptide tag useful for purification, the “HA” tag,corresponds to an epitope derived from the influenza hemagglutininprotein. (Wilson et al., Cell 37:767 (1984).)

Immunophenotyping

The antibodies of the invention may be utilized for immunophenotyping ofcell lines and biological samples. The translation product of the geneof the present invention may be useful as a cell specific marker, ormore specifically as a cellular marker that is differentially expressedat various stages of differentiation and/or maturation of particularcell types. Monoclonal antibodies directed against a specific epitope,or combination of epitopes, will allow for the screening of cellularpopulations expressing the marker. Various techniques can be utilizedusing monoclonal antibodies to screen for cellular populationsexpressing the marker(s), and include magnetic separation usingantibody-coated magnetic beads, “panning” with antibody attached to asolid matrix (i.e., plate), and flow cytometry (See, e.g., U.S. Pat. No.5,985,660; and Morrison et al., Cell, 96:737-49 (1999)).

These techniques allow for the screening of particular populations ofcells, such as might be found with hematological malignancies (i.e.minimal residual disease (MRD) in acute leukemic patients) and“non-self” cells in transplantations to prevent Graft-versus-HostDisease (GVHD). Alternatively, these techniques allow for the screeningof hematopoietic stem and progenitor cells capable of undergoingproliferation and/or differentiation, as might be found in humanumbilical cord blood.

Assays for Antibody Binding

The antibodies of the invention may be assayed for immunospecificbinding by any method known in the art.

Therapeutic Uses

The present invention is further directed to antibody-based therapieswhich involve administering an effective amount of antibodies of thepresent invention to an animal, preferably a mammal, and most preferablya human, patient for treating one or more of the disclosed diseases.disorders, or conditions. Therapeutic compounds of the inventioninclude, but are not limited to, antibodies of the invention (includingfragments, analogs and derivatives thereof as described herein) andnucleic acids encoding antibodies of the invention (including fragments,analogs and derivatives thereof and anti-idiotypic antibodies asdescribed herein). The antibodies of the invention can be used to treat,inhibit or prevent diseases, disorders or conditions associated withaberrant expression and/or activity of a polypeptide of the invention,including, but not limited to, any one or more of the diseases,disorders, or conditions described herein. The treatment and/orprevention of diseases, disorders, or conditions associated withaberrant expression and/or activity of a polypeptide of the inventionincludes, but is not limited to, alleviating symptoms associated withthose diseases, disorders or conditions. Antibodies of the invention maybe provided in pharmaceutically acceptable compositions as known in theart or as described herein.

A summary of the ways in which the antibodies of the present inventionmay be used therapeutically includes binding polynucleotides orpolypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail below.

The antibodies of this invention may be advantageously utilized incombination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors (such as, e.g., KDI, IL-2,IL-3 and IL-7), for example, which serve to increase the number oractivity of effector cells which interact with the antibodies.

The antibodies of the invention may be administered alone or incombination with other types of treatments (e.g., radiation therapy,chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents).Generally, administration of products of a species origin or speciesreactivity (in the case of antibodies) that is the same species as thatof the patient is preferred. Thus, in a preferred embodiment, humanantibodies, fragments derivatives, analogs, or nucleic acids, areadministered to a human patient for therapy or prophylaxis.

It is preferred to use high affinity and/or potent in vivo inhibitingand/or neutralizing antibodies against polypeptides or polynucleotidesof the present invention, fragments or regions thereof, for bothimmunoassays directed to and therapy of disorders related topolynucleotides or polypeptides, including fragments thereof, of thepresent invention. Such antibodies, fragments, or regions, willpreferably have an affinity for polynucleotides or polypeptides of theinvention, including fragments thereof. Preferred binding affinitiesinclude those with a dissociation constant or Kd less than 5×10⁻²M, 10⁻²M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M,10⁻⁶M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M,10⁻¹⁰ M, 5×10⁻¹¹ M, 5×10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M,5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, and 10⁻¹⁵ M.

Methods of Producing Antibodies

The antibodies of the invention can be produced by any method known inthe art for the synthesis of antibodies, in particular, by chemicalsynthesis or preferably, by recombinant expression techniques.

Recombinant expression of an antibody of the invention, or fragment,derivative or analog thereof, (e.g., a heavy or light chain of anantibody of the invention or a single chain antibody of the invention),requires construction of an expression vector containing apolynucleotide that encodes the antibody. Once a polynucleotide encodingan antibody molecule or a heavy or light chain of an antibody, orportion thereof (preferably containing the heavy or light chain variabledomain), of the invention has been obtained, the vector for theproduction of the antibody molecule may be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing a protein by expressing a polynucleotide containing anantibody encoding nucleotide sequence are described herein. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing antibody coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic, recombination. The invention,thus, provides replicable vectors comprising a nucleotide sequenceencoding an antibody molecule of the invention, or a heavy or lightchain thereof, or a heavy or light chain variable domain, operablylinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g., PCTPublication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No.5,122,464) and the variable domain of the antibody may be cloned intosuch a vector for expression of the entire heavy or light chain.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody of the invention. Thus, the inventionincludes host cells containing a polynucleotide encoding an antibody ofthe invention, or a heavy or light chain thereof, or a single chainantibody of the invention, operably linked to a heterologous promoter.In preferred embodiments for the expression of double-chainedantibodies, vectors encoding both the heavy and light chains may beco-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to expressthe antibody molecules of the invention. Such host-expression systemsrepresent vehicles by which the coding sequences of interest may beproduced and subsequently purified, but also represent cells which may,when transformed or transfected with the appropriate nucleotide codingsequences, express an antibody molecule of the invention in situ. Theseinclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing antibody codingsequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5Kpromoter). Preferably, bacterial cells such as Escherichia coli, andmore preferably, eukaryotic cells, especially for the expression ofwhole recombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary cells (CHO), in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2(1990)).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791(1983)), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, NucleicAcids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.24:5503-5509 (1989)); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding tomatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts. (e.g., see Logan &Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specificinitiation signals may also be required for efficient translation ofinserted antibody coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner et al., Methodsin Enzymol. 153:51-544 (1987)).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK,293, 3T3, WI 38, and in particular, breast cancer cell lines such as,for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammarygland cell line such as, for example, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the antibody molecule.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223(1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl.Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991);Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan,Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem.62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215); and hygro, whichconfers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)).Methods commonly known in the art of recombinant DNA technology may beroutinely applied to select the desired recombinant clone, and suchmethods are described, for example, in Ausubel et al. (eds.), CurrentProtocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler,Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY(1990); and in Chapters 12 and 13, Dracopoli et al. (eds), CurrentProtocols in Human Genetics, John Wiley & Sons, NY (1994);Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which areincorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York,1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257(1983)).

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes, and is capable of expressing,both heavy and light chain polypeptides. In such situations, the lightchain should be placed before the heavy chain to avoid an excess oftoxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc.Natl. Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavyand light chains may comprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been produced by ananimal, chemically synthesized, or recombinantly expressed, it may bepurified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigenafter Protein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. In addition, the antibodies of the presentinvention or fragments thereof can be fused to heterologous polypeptidesequences described herein or otherwise known in the art, to facilitatepurification.

The present invention encompasses antibodies recombinantly fused orchemically conjugated (including both covalently and non-covalentlyconjugations) to a polypeptide (or portion thereof, preferably at least10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of thepolypeptide) of the present invention to generate fusion proteins. Thefusion does not necessarily need to be direct, but may occur throughlinker sequences. The antibodies may be specific for antigens other thanpolypeptides (or portion thereof, preferably at least 10, 20, 30, 40,50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the presentinvention. For example, antibodies may be used to target thepolypeptides of the present invention to particular cell types, eitherin vitro or in vivo, by fusing or conjugating the polypeptides of thepresent invention to antibodies specific for particular cell surfacereceptors. Antibodies fused or conjugated to the polypeptides of thepresent invention may also be used in in vitro immunoassays andpurification methods using methods known in the art. See e.g., Harbor etal., supra, and PCT publication WO 93/21232; EP 439,095; Naramura etal., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies etal., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol. 146:2446-2452(1991), which are incorporated by reference in their entireties.

The present invention further includes compositions comprising thepolypeptides of the present invention fused or conjugated to antibodydomains other than the variable regions. For example, the polypeptidesof the present invention may be fused or conjugated to an antibody Fcregion, or portion thereof. The antibody portion fused to a polypeptideof the present invention may comprise the constant region, hinge region,CH1 domain, CH2 domain, and CH3 domain or any combination of wholedomains or portions thereof. The polypeptides may also be fused orconjugated to the above antibody portions to form multimers. Forexample, Fc portions fused to the polypeptides of the present inventioncan form dimers through disulfide bonding between the Fc portions.Higher multimeric forms can be made by fusing the polypeptides toportions of IgA and IgM. Methods for fusing or conjugating thepolypeptides of the present invention to antibody portions are known inthe art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046;5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCTpublications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl.Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. Immunol.154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA89:11337-11341 (1992) (said references incorporated by reference intheir entireties).

As discussed, supra, the polypeptides corresponding to a polypeptide,polypeptide fragment, or a variant of the polypeptides of the presentinvention may be fused or conjugated to the above antibody portions toincrease the in vivo half life of the polypeptides or for use inimmunoassays using methods known in the art. Further, the polypeptidescorresponding to the polypeptides of the present invention may be fusedor conjugated to the above antibody portions to facilitate purification.One reported example describes chimeric proteins consisting of the firsttwo domains of the human CD4-polypeptide and various domains of theconstant regions of the heavy or light chains of mammalianimmunoglobulins. (EP 394,827; Traunecker et al., Nature 331:84-86(1988). The polypeptides of the present invention fused or conjugated toan antibody having disulfide-linked dimeric structures (due to the IgG)may also be more efficient in binding and neutralizing other molecules,than the monomeric secreted protein or protein fragment alone.(Fountoulakis et al., J. Biochem. 270:3958-3964 (1995)). In many cases,the Fc part in a fusion protein is beneficial in therapy and diagnosis,and thus can result in, for example, improved pharmacokineticproperties. (EP A 232,262). Alternatively, deleting the Fc part afterthe fusion protein has been expressed, detected, and purified, would bedesired. For example, the Fc portion may hinder therapy and diagnosis ifthe fusion protein is used as an antigen for immunizations. In drugdiscovery, for example, human proteins, such as hIL-5, have been fusedwith Fc portions for the purpose of high-throughput screening assays toidentify antagonists of hIL-5. (See, Bennett et al., J. MolecularRecognition 8:52-58 (1995); Johanson et al., J. Biol. Chem.270:9459-9471 (1995).

Moreover, the antibodies or fragments thereof of the present inventioncan be fused to marker sequences, such as a peptide to facilitatepurification. In preferred embodiments, the marker amino acid sequenceis a hexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984))and the FLAG tag.

The present invention further encompasses antibodies or fragmentsthereof conjugated to a diagnostic or therapeutic agent. The antibodiescan be used diagnostically to, for example, monitor the development orprogression of a tumor as part of a clinical testing procedure to, e.g.,determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling the antibody to a detectable substance. Examplesof detectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions. Thedetectable substance may be coupled or conjugated either directly to theantibody (or fragment thereof) or indirectly, through an intermediate(such as, for example, a linker known in the art) using techniques knownin the art. See, for example, U.S. Pat. No. 4,741,900 for metal ionswhich can be conjugated to antibodies for use as diagnostics accordingto the present invention. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude 125I, 131I, 111In or 99Tc.

Further, an antibody or fragment thereof may be conjugated to atherapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidalagent, a therapeutic agent or a radioactive metal ion, e.g.,alpha-emitters such as, for example, 213Bi. A cytotoxin or cytotoxicagent includes any agent that is detrimental to cells. Examples includepaclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic agent or drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, a-interferon,.beta.-interferon, nerve growth factor, platelet derived growth factor,tissue plasminogen activator, an apoptotic agent, e.g., TNF-alpha,TNF-beta, lymphotoxin alpha, AIM I (See, International Publication No.WO 97/33899), AIM II (See, International Publication No. WO 97/34911),Fas Ligand (Takahashi et al., Int. Immunol., 6:1567-1574 (1994)), VEGI(See, International Publication No. WO 99/23105), a thrombotic agent oran anti-angiogenic agent, e.g., angiostatin or endostatin; or,biological response modifiers such as, for example, lymphokines,interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”),granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocytecolony stimulating factor (“G-CSF”), or other growth factors.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Amon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-5.6 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. 62:119-58 (1982).

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980, which is incorporated herein by reference in its entirety.

An antibody, with or without a therapeutic moiety conjugated to it,administered alone or in combination with cytotoxic factor(s) and/orcytokine(s) can be used as a therapeutic.

Nucleic Acid Molecules

By “nucleotide sequence” of a nucleic acid molecule or polynucleotide isintended, for a DNA molecule or polynucleotide, a sequence ofdeoxyribonucleotides, and for an RNA molecule or polynucleotide, thecorresponding sequence of ribonucleotides (A, G, C and U), where eachthymidine deoxyribonucleotide (T) in the specified deoxyribonucleotidesequence is replaced by the ribonucleotide uridine (U).

By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its native environmentFor example, recombinant DNA molecules contained in a vector areconsidered isolated for the purposes of the present invention. Furtherexamples of isolated DNA molecules include recombinant DNA moleculesmaintained in heterologous host cells, purified (partially orsubstantially) DNA molecules in solution and synthetic polynucleotides.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe DNA molecules of the present invention. However, a nucleic acidcontained in a clone that is a member of a library (e.g., a genomic orcDNA library) that has not been isolated from other members of thelibrary (e.g., in the form of a homogenous solution containing the cloneand other members of the library) or a chromosome isolated or removedfrom a cell or cell lysate (e.g., a “chromosome spread”, as in akaryotype), is not “isolated” for the purposes of this invention. Asdiscussed further herein, isolated nucleic acid molecules according tothe present invention may be produced naturally, recombinantly orsynthetically.

The stringency of the hybridization and wash depend primarily on thesalt concentration and temperature of the solutions. In general, tomaximize the rate of annealing of the probe with its target, thehybridization is usually carried out at salt and temperature conditionsthat are 20-25° C. below the calculated T_(m) of the of the hybrid. Washconditions should be as stringent as possible for the degree of identityof the probe for the target. In general, wash conditions are selected tobe approximately 12-20° C. below the T_(m) of the hybrid. In regards tothe nucleic acids of the current invention, a moderate stringencyhybridization is defined as hybridization in 6×SSC, 5×Denhardt'ssolution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA at 42° C.,and wash in 2×SSC and 0.5% SDS at 55° C. for 15 minutes. A highstringency hybridization is defined as hybridization in 6×SSC,5×Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNAat 42° C., and wash in 1×SSC and 0.5% SDS at 6-5° C. for 15 minutes.Very high stringency hybridization is defined as hybridization in 6×SSC,S×Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNAat 42° C., and wash in 0.1×SSC and 0.5% SDS at 65° C. for 15 minutes.

As one of ordinary skill would appreciate, due to the possibilities ofsequencing errors discussed above, the actual complete polypeptides ofthe present invention may be somewhat longer or shorter. More generally,the actual open reading frame may be anywhere in the range of ±20 aminoacids, more likely in the range of ±10 amino acids, of that predictedfrom the methionine codon at the N-terminus shown in (SEQ ID NOS: 1, 3,5, 7, 9, 11).

The present invention also relates to the genes of the present inventioncorresponding to SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and/or12. The genes of the present invention can be isolated in accordancewith known methods using the sequence information disclosed herein. Suchmethods include preparing probes or primers from the disclosed sequenceand identifying or amplifying the genes of the present invention fromappropriate sources of genomic material.

Also provided in the present invention are allelic variants, orthologs,and/or species homologs. Procedures known in the art can be used toobtain full-length genes, allelic variants, splice variants, full-lengthcoding portions, orthologs, and/or species homologs of genescorresponding to SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and/or12, using information from the sequences disclosed herein. For example,allelic variants and/or species homologs may be isolated and identifiedby making suitable probes or primers from the sequences provided hereinand screening a suitable nucleic acid source for allelic variants and/orthe desired homologue. See also, for example, U.S. Patent ApplicationPub. No. 2002 039763, which is incorporated herein by reference in itsentirety, including the sequences.

Unless otherwise specified, the term expression vector generally meansany effective nucleic acid having a promoter operably linked to thenucleic acid molecule to be expressed and which in its part may beoperably linked to an effective transcription terminator, preferablysuch that the expression vector effectively expresses the nucleic acidmolecule to be expressed given suitable conditions.

The nucleic acid compositions of the present invention can be preparedin any suitable manner.

The polypeptides encoded by the nucleic acid compositions of the presentinvention may be in the form of the secreted protein, including themature form, or may be a part of a larger protein, such as a fusionprotein. It is often advantageous to include a nucleic acid encoding anadditional amino acid sequence which contains secretory or leadersequences, pro-sequences, sequences which aid in purification, such asmultiple histidine residues, FLAG sequences, or an additional sequencefor stability during recombinant production.

Nucleic acids encoding polypeptides of the present invention,particularly IFN-λs, soluble receptor polypeptides and antibodies of thepresent invention, are preferably provided in an isolated form, andpreferably are substantially purified.

The present invention also provides an isolated nucleic acid moleculecomprising a nucleotide sequence encoding a chimeric receptor comprisingthe extracellular domain of CRF2-12 and the intracellular domain ofanother membrane bound receptor. The present invention also provides anisolated nucleic acid molecule comprising a nucleotide sequence encodinga chimeric receptor comprising the extracellular domain of CRF2-12 andthe intracellular domain of another membrane bound tyrosine kinasereceptor. The present invention also provides an isolated nucleic acidmolecule comprising a nucleotide sequence encoding a chimeric receptorcomprising the extracellular domain of CRF2-12 and the intracellulardomain of a cytokine receptor. The present invention also provides anisolated nucleic acid molecule comprising a nucleotide sequence encodinga chimeric receptor comprising the extracellular domain of CRF2-12 andthe intracellular domain of an IFN R1 type receptor. The presentinvention also provides an isolated nucleic acid molecule comprising anucleotide sequence encoding a chimeric receptor comprising theextracellular domain of CRF2-12 and the intracellular domain of IFNγR1.The present invention also provides an isolated nucleic acid moleculecomprising a nucleotide sequence encoding the chimeric protein inplasmid pEF-CRF2-12/γR1. The present invention also provides an isolatednucleic acid molecule comprising a nucleotide sequence complementary toany of these nucleotide sequences.

The present invention also provides an isolated nucleic acid moleculecomprising a nucleotide sequence encoding a chimeric receptor comprisingthe intracellular domain of CRF2-12 and the extracellular domain ofanother membrane bound receptor. The present invention also provides anisolated nucleic acid molecule comprising a nucleotide sequence encodinga chimeric receptor comprising the intracellular domain of CRF2-12 andthe extracellular domain of another membrane bound tyrosine kinasereceptor. The present invention also provides an isolated nucleic acidmolecule comprising a nucleotide sequence encoding a chimeric receptorcomprising the intracellular domain of CRF2-12 and the extracellulardomain of a cytokine receptor; a nucleotide sequence encoding a chimericreceptor comprising the intracellular domain of CRF2-12 and theextracellular domain of an IFN R1 type receptor. The present inventionalso provides an isolated nucleic acid molecule comprising a nucleotidesequence encoding a chimeric receptor comprising the intracellulardomain of CRF2-12 and the extracellular domain of IL-10R1. The presentinvention also provides an isolated nucleic acid molecule comprising anucleotide sequence encoding the chimeric protein in plasmidpEF-IL-10R1/IFN-λR1. The present invention also provides an isolatednucleic acid molecule comprising a nucleotide sequence complementary toany of these nucleotide sequences.

The present invention also provides an isolated nucleic acid moleculecomprising a tandem vector having a nucleotide sequence encoding tworeceptors, wherein the first receptor is an R1 type receptor and thesecond receptor is an R2 type receptor, and wherein the expression ofeach receptor is controlled by separate promoters and polyadenylationsignals. The present invention also provides an isolated nucleic acidmolecule comprising a tandem vector having a nucleotide sequenceencoding two receptors, wherein the first receptor comprises theextracellular domain of CRF2-12 and the second receptor is an R2 typereceptor, and wherein the expression of each receptor is controlled byseparate promoters and polyadenylation signals. The present inventionalso provides an isolated nucleic acid molecule comprising a tandemvector having a nucleotide sequence encoding two receptors, wherein thefirst receptor comprises the intracellular domain of CRF2-12 and thesecond receptor is an R2 type receptor, and wherein the expression ofeach receptor is controlled by separate promoters and polyadenylationsignal. The present invention also provides an isolated nucleic acidmolecule comprising a tandem vector having a nucleotide sequenceencoding two receptors, wherein the first receptor comprises CRF2-12 andthe second receptor is an R2 type receptor, and wherein the expressionof each receptor is controlled by separate promoters and polyadenylationsignals. The present invention also provides an isolated nucleic acidmolecule comprising any of the above tandem vectors, wherein the R2 typereceptor comprises CRF24. The present invention also provides anisolated nucleic acid molecule comprising the tandem vectorpEF-CRF2-12/γR1+CRF2-4. The present invention also provides an isolatednucleic acid molecule comprising a nucleotide sequence complementary toany of these nucleotide sequences.

The present invention also provides isolated nucleic acid moleculescomprising a polynucleotide encoding at least a portion of one of theIFN-λ polypeptides having the complete amino acid sequence shown of SEQID NOS: 8, 10 and 12.

The nucleotide sequence determined by sequencing the IFN-λ1 clone, whichis shown in FIG. 16 (SEQ ID NO: 7), contains an open reading frameencoding a full length polypeptide of 200 amino acid residues, includingan initiation codon encoding an N-terminal methionine.

The nucleotide sequence determined by sequencing the IFN-λ2 clone, whichis shown in FIG. 17 (SEQ ID NO: 9), contains an open reading frameencoding a full length polypeptide of 196 amino acid residues, includingan initiation codon encoding an N-terminal methionine.

The nucleotide sequence determined by sequencing the IFN-λ3 clone, whichis shown in FIG. 18 (SEQ ID NO: 11), contains an open reading frameencoding a full length polypeptide of 196 amino acid residues, includingan initiation codon encoding an N-terminal methionine.

The present invention also provides isolated nucleic acid moleculesencoding the complete amino acid sequence excepting the N-terminalmethionine shown in SEQ ID NOS: 8, 10 and 12, which molecules also canencode additional amino acids fused to the N-terminus of the amino acidsequences of IFN-λ1, IFN-λ2 and/or IFN-λ3.

Another aspect of the invention provides an isolated nucleic acidmolecule comprising a nucleotide sequence encoding the IFN-λ1polypeptide having the complete amino acid sequence of SEQ ID NO: 8.Another aspect of the invention provides an isolated nucleic acidmolecule comprising a nucleotide sequence encoding the IFN-λ1polypeptide having the complete amino acid sequence of SEQ ID NO: 8excepting the N-terminal methionine (i.e., residues 2-200 of SEQ ID NO:8). Another aspect of the invention provides an isolated nucleic acidmolecule comprising a nucleotide sequence encoding the mature IFN-λ1polypeptide shown as residues 23-200 of SEQ ID NO: 8. Another aspect ofthe invention provides an isolated nucleic acid molecule comprising anucleotide sequence of the genomic fragment encoding the complete IFN-λ1gene contained in plasmid pEF-FL-IFN-λ1 gene. Another aspect of theinvention provides an isolated nucleic acid molecule comprising anucleotide sequence encoding the complete polypeptide encoded by thecDNA contained in plasmid pEF-FL-IFN-λ1; a nucleotide sequence encodingthe mature polypeptide encoded by the human cDNA contained in plasmidpEF-FL-IFN-λ1. Another aspect of the invention provides an isolatednucleic acid molecule comprising a nucleotide sequence complementary toany of these nucleotide sequences.

Another aspect of the invention provides an isolated nucleic acidmolecule comprising a nucleotide sequence encoding the IFN-λ2polypeptide having the complete amino acid sequence of SEQ ID NO: 10.Another aspect of the invention provides an isolated nucleic acidmolecule comprising a nucleotide sequence encoding the IFN-λ2polypeptide having the complete amino acid sequence of SEQ ID NO: 10excepting the N-terminal methionine (i.e., residues 2-196 of SEQ ID NO:10). Another aspect of the invention provides an isolated nucleic acidmolecule comprising a nucleotide sequence encoding the mature IFN-λ2polypeptide shown as residues 23-196 of SEQ ID NO: 10. Another aspect ofthe invention provides an isolated nucleic acid molecule comprising anucleotide sequence encoding IFN-λ2 polypeptide (residues M12-196 of SEQID NO: 10). Another aspect of the invention provides an isolated nucleicacid molecule comprising a nucleotide sequence encoding IFN-λ2polypeptide residues A13-196 of SEQ ID NO: 10. Another aspect of theinvention provides an isolated nucleic acid molecule comprising anucleotide sequence encoding the mature IFN-λ2 polypeptide shown asresidues P31-196 of SEQ ID NO: 10. Another aspect of the inventionprovides an isolated nucleic acid molecule comprising a nucleotidesequence encoding IFN-λ2 polypeptide (residues M92-196 of SEQ ID NO:10). Another aspect of the invention provides an isolated nucleic acidmolecule comprising a nucleotide sequence encoding IFN-λ2 polypeptideresidues A93-196 of SEQ ID NO: 10. Another aspect of the inventionprovides an isolated nucleic acid molecule comprising a nucleotidesequence encoding the mature IFN-λ2 polypeptide shown as residuesP113-196 of SEQ ID NO: 10. Another aspect of the invention provides anisolated nucleic acid molecule comprising a nucleotide sequence of thegenomic fragment encoding the complete IFN-2 gene contained in plasmidpEF-FL-IFN-λ2gene. Another aspect of the invention provides an isolatednucleic acid molecule comprising a nucleotide sequence encoding apolypeptide encoded by the cDNA contained in plasmid pEF-FL-IFN-λ2.Another aspect of the invention provides an isolated nucleic acidmolecule comprising a nucleotide sequence encoding the maturepolypeptide encoded by the cDNA contained in clone pEF-FL-IFN-λ2.Another aspect of the invention provides an isolated nucleic acidmolecule comprising a nucleotide sequence complementary to any of thesenucleotide sequences.

Another aspect of the invention provides an isolated nucleic acidmolecule comprising a nucleotide sequence encoding the IFN-λ3polypeptide having the complete amino acid sequence of SEQ ID NO: 12.Another aspect of the invention provides an isolated nucleic acidmolecule comprising a nucleotide sequence encoding the IFN-λ3polypeptide having the complete amino acid sequence of SEQ ID NO: 12excepting the N-terminal methionine (i.e., residues 2-196 of SEQ ID NO:12). Another aspect of the invention provides an isolated nucleic acidmolecule comprising a nucleotide sequence encoding the mature IFN-λ3polypeptide shown as residues P23-196 of SEQ ID NO: 12. Another aspectof the invention provides an isolated nucleic acid molecule comprising anucleotide sequence encoding IFN-λ3 polypeptide (residues M6-196 of SEQID NO: 12). Another aspect of the invention provides an isolated nucleicacid molecule comprising a nucleotide sequence encoding IFN-λ3polypeptide (residues M12-196 of SEQ ID NO: 12). Another aspect of theinvention provides an isolated nucleic acid molecule comprising anucleotide sequence encoding IFN-λ3 polypeptide residues P7-196 of SEQID NO: 12. Another aspect of the invention provides an isolated nucleicacid molecule comprising a nucleotide sequence encoding IFN-λ3polypeptide residues A13-196 of SEQ ID NO: 12. Another aspect of theinvention provides an isolated nucleic acid molecule comprising anucleotide sequence encoding the mature IFN-λ3 polypeptide shown asresidues P31-196 of SEQ ID NO: 12. Another aspect of the inventionprovides an isolated nucleic acid molecule comprising a nucleotidesequence of the genomic fragment encoding the complete IFN-λ3 genecontained in plasmid pEF-FL-IFN-λ3gene. Another aspect of the inventionprovides an isolated nucleic acid molecule comprising a nucleotidesequence encoding a polypeptide encoded by the cDNA contained in plasmidpEF-FL-IFN-λ3. Another aspect of the invention provides an isolatednucleic acid molecule comprising a nucleotide sequence encoding a maturepolypeptide encoded by the cDNA contained in plasmid pEF-FL-IFN-λ3.Another aspect of the invention provides an isolated nucleic acidmolecule comprising a nucleotide sequence complementary to any of thesenucleotide sequences.

The invention also provides isolated nucleic acid molecules comprising apolynucleotide encoding at least a portion of one of the IFN-λpolypeptides described in the “Polypeptides” section of the presentdisclosure.

Further embodiments of the invention include isolated nucleic acidmolecules that comprise a polynucleotide having a nucleotide sequence atleast 80%, 85%, or 90% identical, more preferably at least 91%, 92%,93%, and 94% and most preferably at least 95%, 96%, 97%, 98% or to anyof the nucleotide sequences, above, or a polynucleotide which hybridizesunder stringent hybridization conditions to a polynucleotide of thepresent invention. This polynucleotide of the present invention, whichhybridizes under stringent conditions does not hybridize to apolynucleotide having a nucleotide sequence consisting of only Aresidues or of only T residues. An additional nucleic acid embodiment ofthe invention relates to an isolated nucleic acid molecule comprising apolynucleotide that encodes the amino acid sequence of anepitope-bearing portion of a polypeptide having an amino acid sequencedescribed above.

A further aspect of the invention is a DNA sequence that represents thecomplete upstream and downstream regulatory regions of the genes of thepresent invention. DNA constructs containing the regulatory region arealso provided. Further, host cells comprising such constructs, whichcells are in vitro or in vivo, are also encompassed by the presentinvention.

The present invention also relates to recombinant vectors, which includethe isolated nucleic acid molecules of the present invention, to hostcells containing the recombinant vectors, as well as to methods ofmaking such vectors and host cells and for using them for production ofpolypeptides or peptides of the present invention by recombinanttechniques.

Leader and Mature Sequences

The amino acid sequence of the complete proteins of the presentinvention includes a leader sequence and a mature protein, as shown inFIGS. 14A and 19A. Accordingly, the present invention provides nucleicacid molecules encoding a mature form of the proteins of the presentinvention having the polypeptide sequence of SEQ ID NOS:2, 6, 8, 10and/or 12. Polynucleotides encoding the mature forms are alsoencompassed by the invention.

According to the current understanding in the art, once export of agrowing protein chain across the rough endoplasmic reticulum has beeninitiated, proteins secreted by mammalian cells have the signal orsecretory leader sequence cleaved from the complete polypeptide toproduce a secreted “mature” form of the protein. In some instances,proteins having a signal or leader sequence may be retainedintracellularly or at the cell surface. Most mammalian cells and eveninsect cells cleave secreted proteins with the same specificity.However, in some cases, cleavage of a secreted protein is not entirelyuniform, which results in two or more mature species of the protein.Further, it has long been known that the cleavage specificity of asecreted protein is ultimately determined by the primary structure ofthe complete protein, that is, it is inherent in the amino acid sequenceof the polypeptide. Therefore, the present invention provides anucleotide sequences encoding mature polypeptides of the presentinvention having the amino acid sequence encoded by the human cDNA inclones pEF-FL-IFN-λ1, pEF-FL-IFN-λ2 and/or pEF-FL-IFN-λ3 and genomicfragments encoded by pEF-FL-IFN-λ1 gene, pEF-FL-IFN-λ2gene and/orpEF-FL-IFN-λ3gene. By the “mature polypeptides” of the present inventionhaving the amino acid sequence encoded by the human cDNA in clonespEF-FL-IFN-λ1, pEF-FL-IFN-λ2 and/or pEF-FL-IFN-λ3 and genomic fragmentsencoded by pEF-FL-IFN-λ1gene, pEF-FL-IFN-λ2gene and/or pEF-FL-IFN-λ3geneis meant the mature form(s) of the proteins of the present inventionproduced by expression in a mammalian cell (e.g., COS cells, asdescribed below) from the open reading frame encoded by the human DNAsequence of the clone contained in the vector or a portion of the DNAsequence of the clone contained in the vector fused to a heterologoussignal sequence.

Methods for predicting whether a protein has a secretory leader as wellas the cleavage point for that leader sequence are available. Forinstance, the method of McGeoch (Virus Res. 3:271-286 (1985)) uses theinformation from a short N-terminal charged region and a subsequentuncharged region of the complete (uncleaved) protein. The method of vonHeinje (Nucleic Acids Res. 14:4683-4690 (1986)) uses the informationfrom the residues surrounding the cleavage site, typically residues −13to +2 where +1 indicates the amino terminus of the mature protein. Theaccuracy of predicting the cleavage points of known mammalian secretoryproteins for each of these methods is in the range of 75-80% (vonHeinje, supra). However, the two methods do not always produce the samepredicted cleavage point(s) for a given protein.

As indicated, nucleic acid molecules of the present invention may be inthe form of RNA, or in the form of DNA. The DNA may be double-strandedor single-stranded. Single-stranded DNA or RNA may be the coding strand,also known as the sense strand, or it may be the non-coding strand, alsoreferred to as the anti-sense strand.

In a further embodiment, polynucleotides of the invention comprise aportion of the coding sequences. In another embodiment, thepolynucleotides comprising coding sequences do not contain codingsequences of a genomic flanking gene (i.e., 5′ or 3′ to the genes of thepresent invention of interest in the genome).

In addition, isolated nucleic acid molecules of the invention includeDNA molecules which comprise a sequence substantially different fromthose described above but which, due to the degeneracy of the geneticcode, still encodes a polypeptides of the present invention of thepresent invention. Of course, the genetic code and species-specificcodon preferences are well known in the art. Thus, it would be routinefor one skilled in the art to generate the degenerate variants describedabove, for instance, to optimize codon expression for a particular host(e.g., change codons in the human mRNA to those preferred by a bacterialhost such as E. coli, yeast, bacillus, plants, or any other effectivehost).

Treatment

The IFN-λ compositions of the present invention have been shown toactivate the CRF2-12 receptor and receptor complex of the presentinvention, activating many of the same signal-transduction pathwayelements as treatment with IFN-alpha. However the CRF2-12 receptor andreceptor complex of the present invention is not responsive to IFN-alphaor beta. Accordingly, IFN-λ compositions of the present invention shouldhave a clinical advantage to treatment with other interferons. One ofthese advantages derives from the widespread presence of receptorsresponsive to IFN-λs, particularly since there is an area of non-overlapbetween the IFN-alpha/beta receptor complex distribution and the IFN-λreceptor complex distribution. Accordingly, the compositions of thepresent invention may encounter greater clinically efficacy for certaintherapies, known in the art, for which this family of cytokines has beenfound to be useful. Some of these uses are listed in U.S. Pat. No.6,472,512, which is incorporated herein by reference in its entirety.

Thus, the invention provides a method of treatment of an individual inneed of an increased level of interferon activity comprisingadministering to such an individual a pharmaceutical composition of thepresent invention in an amount effective to increase the interferonactivity level in such an individual.

Compositions of the invention may also be useful in treating diseases,disorders, and/or conditions of the immune system. Therefore, it will beappreciated that conditions caused by a decrease in the standard ornormal level of interferon activity in an individual, particularlydisorders of the immune system, can be treated by administration ofpolynucleotides, polypeptides, or agonists or antagonists of thecompositions of the present invention. Thus, the invention also providesa method of treatment of an individual in need of an increased level ofinterferon activity comprising administering to such an individual apharmaceutical composition comprising a therapeutic amount ofpolynucleotides, polypeptides, or agonists or antagonists ofcompositions of the present invention, effective to increase theinterferon activity level in such an individual.

Particularly preferred is treatment of individuals that, for any reason,but preferably one unrelated to the effectiveness of IFN-λ, areunresponsive to IFN-alpha, IFN-beta, IFN-omega, KDI, and other cytokinesor interferons.

Particularly preferred are conditions that may be treated, prevented,ameliorated or diagnosed through the biological effects of a compositionof the present invention. These include, for example, conditionsresponsive to modulation of the JAK-Stat pathway; conditions responsiveto modulation of the ISFG-3 complex; and other such activities modulatedby receptors responsive to polypeptides and fragments of the presentinvention, preferably CRF2-12 and the CRF2-12 receptor complex.

In another embodiment, the allelic type of an individual may bedetermined for a gene or combination of genes of the present invention.The allelic type may be used in the treatment, prevention, ameliorationor diagnosis of disorders related to the compositions of the presentinvention. For example, it may be determined that an individual has anallelic type that is non-functional. In such case, administration of afunctional composition in accordance with the present invention would beeffective in the treatment of the diagnosed condition. In anotherembodiment, the allelic type is determined prior to administration of acomposition of the present invention, and the allelic type administeredis matched to the allelic type of the individual to which thecomposition is to be administered. In such manner, immune reactions maybe avoided against the composition, or the incidence of such occurrencesdecreased.

The allelic type of an individual may be determined in any effectivemanner. For example, the following techniques may be useful in suchdetermination: immunotyping of material from the individual, measurementof biological effects of material obtained from the individual, PCR ofgenomic or other effective nucleic acid from the individual, RT-PCR ofmRNA or other effective nucleic acid from the individual (e.g., from abiopsy, or the like), RFLP, repeat polymorphism linkage (ca repeats, orthe like), sequencing, hybridization, or any other effective technique.

Compositions of the present invention may be used clinically for anyanti-viral therapy for which they are effective. For example, in thetreatment of AIDS, viral hepatitis including chronic hepatitis B,hepatitis C, papilloma viruses (e.g., condyloma acuminatum, laryngealpapillomatosis), viral encephalitis, and in the prophylaxis of rhinitisand respiratory infections.

In a specific embodiment, compositions of the present invention areuseful in the treatment, prevention, detection and/or diagnosis of anunregulated growth, e.g., a cancer, some examples would include hairycell leukemia, bladder carcinoma, cervical carcinoma, acute myeloidleukemia, osteosarcoma, basal cell carcinoma, glioma, renal cellcarcinoma, multiple myeloma, melanoma, and Hodgkin's disease.

Compositions of the present invention are believed to be capable ofstimulating natural killer cell activity. Accordingly, they may be usedto treat any infection for which they are effective. Among theconditions that may find useful treatment by the present invention areCryptosporidium parvum infection and multidrug-resistant pulmonarytuberculosis.

Compositions of the present invention are also believed to be useful asimmunotherapeutic agents. Depending on whether an agonist or antagonistis used, the effect the compositions of the present invention may finduse as immunosuppressive or immunoenhancing agents.

Because of their immunomodulatory function, compositions of the presentinvention may find use as a protective agents when administered prior tochemotherapy. They may also find use in treatment of hyperproliferationof lymphocytes, myeloid progenitors and bone marrow stem cells, asoccurs, for example, in chronic myelogenous leukemia.

The compositions of the present invention may also find use in theprevention of graft vs. host rejection, or to curtail the progression ofautoimmune diseases, such as arthritis, multiple sclerosis, systemiclupus or diabetes. The polynucleotides, polypeptides, or agonists orantagonists of the present invention are also useful in the treatment ofallergies in mammals, e.g., by inhibiting the humoral response.

Compositions of the present invention may be used as an adjuvant orcoadjuvant to enhance or stimulate the immune response in cases ofprophylactic or therapeutic vaccination.

Further, there is provided a method of treating infection in a patientcomprising administering an effective amount of a composition of thepresent invention to a patient in need of anti-infective therapy. In apreferred embodiment the infection is of viral, bacterial, or parasiticetiology. In a particularly preferred embodiment, the infection is aviral infection.

Further, there is provided a method of treating cancer in a patientcomprising administering an effective amount of a composition of thepresent invention to a patient in need of anti-cancer therapy.

Further, there is provided a method of immunotherapy in a patientcomprising administering an effective amount of a composition of thepresent invention to a patient in need of immunotherapy.

Compositions of the present invention, may be useful in treatingdiseases, disorders, and/or conditions of the immune system, byactivating or inhibiting the proliferation, differentiation, ormobilization (chemotaxis) of immune cells. Immune cells develop througha process called hematopoiesis, producing myeloid (platelets, red bloodcells, neutrophils, and macrophages) and lymphoid (B and T lymphocytes)cells from pluripotent stem cells. The etiology of these immunediseases, disorders, and/or conditions may be genetic, somatic, such ascancer or some autoimmune diseases, disorders, and/or conditions,acquired (e.g., by chemotherapy or toxins), or infectious. Moreover,compositions of the present invention, can be used as a marker ordetector of a particular immune system disease or disorder.

Compositions of the present invention may be useful in treating,preventing, and/or diagnosing diseases, disorders, and/or conditions ofhematopoietic cells. Compositions of the present invention could be usedto increase differentiation and proliferation of hematopoietic cells,including the pluripotent stem cells, in an effort to treat or preventthose diseases, disorders, and/or conditions associated with a decreasein certain (or many) types hematopoietic cells. Examples of immunologicdeficiency syndromes include, but are not limited to: blood proteindiseases, disorders, and/or conditions (e.g. agammaglobulinemia,dysgammaglobulinemia), ataxia telangiectasia, common variableimmunodeficiency, Digeorge Syndrome, HIV infection, HTLV-BLV infection,leukocyte adhesion deficiency syndrome, lymphopenia, phagocytebactericidal dysfunction, severe combined immunodeficiency (SCIDs),Wiskott-Aldrich Disorder, anemia, thrombocytopenia, or hemoglobinuria.

Moreover, compositions of the present invention can also be used tomodulate hemostatic (the stopping of bleeding) or thrombolytic activity(clot formation). For example, by increasing hemostatic or thrombolyticactivity, Compositions of the present invention could be used to treator prevent blood coagulation diseases, disorders, and/or conditions(e.g., afibrinogenemia, factor deficiencies), blood platelet diseases,disorders, and/or conditions (e.g. thrombocytopenia), or woundsresulting from trauma, surgery, or other causes. Alternatively,Compositions of the present invention that can decrease hemostatic orthrombolytic activity could be used to inhibit or dissolve clotting.These molecules could be important in the treatment or prevention ofheart attacks (infarction), strokes, or scarring.

Compositions of the present invention may also be useful in treating,preventing, and/or diagnosing autoimmune diseases, disorders, and/orconditions. Many autoimmune diseases, disorders, and/or conditionsresult from inappropriate recognition of self as foreign material byimmune cells. This inappropriate recognition results in an immuneresponse leading to the destruction of the host tissue. Therefore, theadministration of compositions of the present invention that can inhibitan immune response, particularly the proliferation, differentiation, orchemotaxis of T-cells, may be an effective therapy in preventingautoimmune diseases, disorders, and/or conditions.

Examples of autoimmune diseases, disorders, and/or conditions that canbe treated, prevented, and/or diagnosed or detected by the compositionsof the present invention include, but are not limited to: Addison'sDisease, hemolytic anemia, antiphospholipid syndrome, rheumatoidarthritis, dermatitis, allergic encephalomyelitis, glomerulonephritis,Goodpasture's Syndrome, Graves' Disease, Multiple Sclerosis, MyastheniaGravis, Neuritis, Ophthalmia, Bullous Pemphigoid, Pemphigus,Polyendocrinopathies, Purpura, Reiter's Disease, Stiff-Man Syndrome,Autoimmune Thyroiditis, Systemic Lupus Erythematosus, AutoimmunePulmonary Inflammation, Guillain-Barre Syndrome, insulin dependentdiabetes mellitis, and autoimmune inflammatory eye disease.

Similarly, allergic reactions and conditions, such as asthma(particularly allergic asthma) or other respiratory problems, may alsobe treated, prevented, and/or diagnosed by compositions of the presentinvention. Moreover, these molecules can be used to treat anaphylaxis,hypersensitivity to an antigenic molecule, or blood groupincompatibility.

Compositions of the present invention may also be used to treat,prevent, and/or diagnose organ rejection or graft-versus-host disease(GVHD). Organ rejection occurs by host immune cell destruction of thetransplanted tissue through an immune response. Similarly, an immuneresponse is also involved in GVHD, but, in this case, the foreigntransplanted immune cells destroy the host tissues. The administrationof compositions of the present invention that inhibits an immuneresponse, particularly the proliferation, differentiation, or chemotaxisof T-cells, may be an effective therapy in preventing organ rejection orGVHD.

Similarly, compositions of the present invention may also be used tomodulate inflammation. For example, compositions of the presentinvention may inhibit the proliferation and differentiation of cellsinvolved in an inflammatory response. These molecules can be used totreat, prevent, and/or diagnose inflammatory conditions, both chronicand acute conditions, including chronic prostatitis, granulomatousprostatitis and malacoplakia, inflammation associated with infection(e.g., septic shock, sepsis, or systemic inflammatory response syndrome(SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis,complement-mediated hyperacute rejection, nephritis, cytokine orchemokine induced lung injury, inflammatory bowel disease, Crohn'sdisease, or resulting from over production of cytokines (e.g., TNF orIL-1.)

Hyperproliferative Disorders

Compositions of the present invention can be used to treat, prevent,and/or diagnose hyperproliferative diseases, disorders, and/orconditions, including neoplasms. Compositions of the present inventionmay inhibit the proliferation of the disorder through direct or indirectinteractions. Alternatively, compositions of the present invention mayproliferate other cells which can inhibit the hyperproliferativedisorder.

For example, by increasing an immune response, particularly increasingantigenic qualities of the hyperproliferative disorder or byproliferating, differentiating, or mobilizing T-cells,hyperproliferative diseases, disorders, and/or conditions can betreated, prevented, and/or diagnosed. This immune response may beincreased by either enhancing an existing immune response, or byinitiating a new immune response. Alternatively, decreasing an immuneresponse may also be a method of treating, preventing, and/or diagnosinghyperproliferative diseases, disorders, and/or conditions, such as achemotherapeutic agent.

Examples of hyperproliferative diseases, disorders, and/or conditionsthat can be treated, prevented, and/or diagnosed by using compositionsof the present invention include, but are not limited to neoplasmslocated in the: colon, abdomen, bone, breast, digestive system, liver,pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary,testicles, ovary, thymus, thyroid), eye, head and neck, nervous (centraland peripheral), lymphatic system, pelvic, skin, soft tissue, spleen,thoracic, and urogenital.

Similarly, other hyperproliferative diseases, disorders, and/orconditions can also be treated, prevented, and/or diagnosed bycompositions of the present invention. Examples of suchhyperproliferative diseases, disorders, and/or conditions include, butare not limited to: hypergammaglobulinemia, lymphoproliferativediseases, disorders, and/or conditions, paraproteinemias, purpura,sarcoidosis, Sezary Syndrome, Waldenstron's Macroglobulinemia, Gaucher'sDisease, histiocytosis, and any other hyperproliferative disease,besides neoplasia, located in an organ system listed above.

One preferred embodiment utilizes polynucleotides of the presentinvention to inhibit aberrant cellular division, by gene therapy usingthe present invention, and/or protein fusions or fragments thereof.

Thus, the present invention provides a method for treating cellproliferative diseases, disorders, and/or conditions by inserting intoan abnormally proliferating cell a polynucleotide of the presentinvention, wherein said polynucleotide represses said expression.

Another embodiment of the present invention provides a method oftreating cell-proliferative diseases, disorders, and/or conditions inindividuals comprising administration of one or more active gene copiesof the present invention to an abnormally proliferating cell or cells.In a preferred embodiment, polynucleotides of the present invention is aDNA construct comprising a recombinant expression vector effective inexpressing a DNA sequence encoding said polynucleotides. In anotherpreferred embodiment of the present invention, the DNA constructencoding a polynucleotide of the present invention is inserted intocells to be treated utilizing a retrovirus, or more preferrably anadenoviral vector (See G J. Nabel, et. al., PNAS 1999 96: 324-326, whichis hereby incorporated by reference). In a most preferred embodiment,the viral vector is defective and will not transform non-proliferatingcells, only proliferating cells. Moreover, in a preferred embodiment,the polynucleotides of the present invention inserted into proliferatingcells either alone, or in combination with or fused to otherpolynucleotides, can then be modulated via an external stimulus (i.e.magnetic, specific small molecule, chemical, or drug administration,etc.), which acts upon the promoter upstream of said polynucleotides toinduce expression of the encoded protein product. As such the beneficialtherapeutic affect of the present invention may be expressly modulated(i.e. to increase, decrease, or inhibit expression of the presentinvention) based upon said external stimulus.

Polynucleotides of the present invention may be useful in repressingexpression of oncogenic genes or antigens. By “repressing expression ofthe oncogenic genes” is intended the suppression of the transcription ofthe gene, the degradation of the gene transcript (pre-message RNA), theinhibition of splicing, the destruction of the messenger RNA, theprevention of the post-translational modifications of the protein, thedestruction of the protein, or the inhibition of the normal function ofthe protein.

For local administration to abnormally proliferating cells,polynucleotides of the present invention may be administered by anymethod known to those of skill in the art including, but not limited totransfection, electroporation, microinjection of cells, or in vehiclessuch as liposomes, lipofectin, or as naked polynucleotides, or any othermethod described throughout the specification. The polynucleotide of thepresent invention may be delivered by known gene delivery systems suchas, but not limited to, retroviral vectors (Gilboa, J. Virology 44:845(1982); Hocke, Nature 320:275 (1986); Wilson, et al., Proc. Natl. Acad.Sci. U.S.A. 85:3014), vaccinia virus system (Chakrabarty et al., Mol.Cell. Biol. 5:3403 (1985) or other efficient DNA delivery systems (Yateset al., Nature 313:812 (1985)) known to those skilled in the art. Thesereferences are exemplary only and are hereby incorporated by reference.In order to specifically deliver or transfect cells which are abnormallyproliferating and spare non-dividing cells, it is preferable to utilizea retrovirus, or adenoviral (as described in the art and elsewhereherein) delivery system known to those of skill in the art. Since hostDNA replication is required for retroviral DNA to integrate and theretrovirus will be unable to self replicate due to the lack of theretrovirus genes needed for its life cycle. Utilizing such a retroviraldelivery system for polynucleotides of the present invention will targetsaid gene and constructs to abnormally proliferating cells and willspare the non-dividing normal cells.

The polynucleotides of the present invention may be delivered directlyto cell proliferative disorder/disease sites in internal organs, bodycavities and the like by use of imaging devices used to guide aninjecting needle directly to the disease site. The polynucleotides ofthe present invention may also be administered to disease sites at thetime of surgical intervention.

By “cell proliferative disease” is meant any human or animal disease ordisorder, affecting any one or any combination of organs, cavities, orbody parts, which is characterized by single or multiple local abnormalproliferations of cells, groups of cells, or tissues, whether benign ormalignant.

Any amount of the polynucleotides of the present invention may beadministered as long as it has a biologically inhibiting effect on theproliferation of the treated cells. Moreover, it is possible toadminister more than one of the polynucleotide of the present inventionconcurrently to the same site. By “biologically inhibiting” is meantpartial or total growth inhibition as well as decreases in the rate ofproliferation or growth of the cells. The biologically inhibitory dosemay be determined by assessing the effects of the polynucleotides of thepresent invention on target malignant or abnormally proliferating cellgrowth in tissue culture, tumor growth in animals and cell cultures, orany other method known to one of ordinary skill in the art.

As used herein, the term “concurrently” has the same meaning as itsdefinition of U.S. Pat. No. 6,248,725, which is incorporated herein byreference in its entirety.

The present invention is further directed to antibody-based therapieswhich involve administering of anti-polypeptides and anti-polynucleotideantibodies to a mammalian, preferably human, patient for treating one ormore of the described diseases, disorders, and/or conditions. Methodsfor producing anti-polypeptides and anti-polynucleotide antibodiespolyclonal and monoclonal antibodies are described in detail elsewhereherein. Such antibodies may be provided in pharmaceutically acceptablecompositions as known in the art or as described herein.

A summary of the ways in which the antibodies of the present inventionmay be used therapeutically includes binding polynucleotides orpolypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail herein. Those of ordinary skill in the artwill know how to use the antibodies of the present invention fordiagnostic, monitoring or therapeutic purposes without undueexperimentation.

In particular, the antibodies, fragments and derivatives of the presentinvention are useful for treating a subject having or developing cellproliferative and/or differentiation diseases, disorders, and/orconditions as described herein. Such treatment comprises administering asingle or multiple doses of the antibody, or a fragment, derivative, ora conjugate thereof.

The antibodies of this invention may be advantageously utilized incombination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors, for example, which serve toincrease the number or activity of effector cells which interact withthe antibodies.

It is preferred to use high affinity and/or potent in vivo inhibitingand/or neutralizing antibodies against a composition of the presentinvention, fragments or regions thereof, for both immunoassays andtherapy of diseases, disorders, and/or conditions related tocompositions of the present invention. Such antibodies, fragments, orregions, will preferably have an affinity for polynucleotides orpolypeptides, including fragments thereof. Preferred binding affinitiesinclude those with a dissociation constant or Kd less than 5×10⁻⁶ M,10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M,10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M,5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, and 10⁻¹⁵ M.

Moreover, polypeptides of the present invention may find one inhibitingthe angiogenesis of proliferative cells or tissues, either alone, as aprotein fusion, or in combination with other polypeptides directly orindirectly, as described elsewhere herein. In a most preferredembodiment, said anti-angiogenesis effect may be achieved indirectly,for example, through the inhibition of hematopoietic, tumor-specificcells, such as tumor-associated macrophages (See Joseph I B, et al. JNatl Cancer Inst, 90(21):1648-53 (1998), which is hereby incorporated byreference). Antibodies directed to compositions of the present inventionmay also result in inhibition of angiogenesis directly, or indirectly(See Witte L, et al., Cancer Metastasis Rev. 17(2):155-61 (1998), whichis hereby incorporated by reference)).

Compositions of the present invention may be useful in inhibitingproliferative cells or tissues through the induction of apoptosis. Saidcompositions may act either directly, or indirectly to induce apoptosisof proliferative cells and tissues. Moreover, in another preferredembodiment of the present invention, said compositions, either alone orin combination with small molecule drugs or adjuvants, such asapoptonin, galectins, thioredoxins, antiinflammatory proteins may induceapoptosis through other mechanisms, such as in the activation of otherproteins which will activate apoptosis, or through stimulating theexpression of said proteins, (See for example, Mutat Res 400(1-2):447-55(1998), Med. Hypotheses. 50(5):423-33 (1998) Biol Interact. April 24;111-112:23-34 (1998), J Mol. Med. 76(6):402-12 (1998), Int J TissueReact; 20(1):3-15 (1998), which are all hereby incorporated byreference).

Compositions of the present invention are useful in inhibiting themetastasis of proliferative cells or tissues. Such therapeutic affectsof the present invention may be achieved either alone, or in combinationwith small molecule drugs or adjuvants.

Compositions of the present invention are useful in enhancing theimmunogenicity and/or antigenicity of proliferating cells or tissues,either directly, such as would occur if the polypeptides of the presentinvention ‘vaccinated’ the immune response to respond to proliferativeantigens and immunogens, or indirectly, such as in activating theexpression of proteins known to enhance the immune response (e.g.chemokines), to said antigens and immunogens.

Cardiovascular Disorders

Nucleic acids of the present invention may be used to treat, prevent,and/or diagnose cardiovascular diseases, disorders, and/or conditions,including peripheral artery disease, such as limb ischemia. Otherdisorders listed in U.S. Pat. No. 6,472,512.

The present invention provides for treatment of diseases, disorders,and/or conditions associated with neovascularization by administrationof a composition of the present invention. Malignant and metastaticconditions which can be treated with the polynucleotides andpolypeptides, or agonists or antagonists of the invention include, butare not limited to, malignancies, solid tumors, and cancers describedherein and otherwise known in the art (for a review of such disorders,see Fishman et al., Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia(1985)). Thus, the present invention provides a method of treating anangiogenesis-related disease and/or disorder, comprising administeringto an individual in need thereof a therapeutically effective amount of acomposition of the invention. For example, polynucleotides,polypeptides, antagonists and/or agonists may be utilized in a varietyof additional methods in order to therapeutically treat or prevent acancer or tumor. Cancers which may be treated or diagnosed withcompositions of the present invention are listed in U.S. Pat. No.6,472,512, which is incorporated herein by reference in its entirety.

Within particularly preferred embodiments of the invention, may beprepared for topical administration in saline (combined with any of thepreservatives and antimicrobial agents commonly used in ocularpreparations), and administered in eyedrop form. The solution orsuspension may be prepared in its pure form and administered severaltimes daily.

The compositions of the present invention may also be administered alongwith other anti-angiogenic factors. Representative examples of otheranti-angiogenic factors include: Anti-invasive Factor, retinoic acid andderivatives thereof, paclitaxel, Suramin, Tissue Inhibitor ofMetalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2,Plasminogen Activator Inhibitor-1, Plasminogen Activator Inhibitor-2,and various forms of the lighter “d group” transition metals.

Lighter “d group” transition metals include, for example, vanadium,molybdenum, tungsten, titanium, niobium, and tantalum species. Suchtransition metal species may form transition metal complexes. Suitablecomplexes of the above-mentioned transition metal species include oxotransition metal complexes.

Representative examples of vanadium complexes include oxo vanadiumcomplexes such as vanadate and vanadyl complexes. Suitable vanadatecomplexes include metavanadate and orthovanadate complexes such as, forexample, ammonium metavanadate, sodium metavanadate, and sodiumorthovanadate. Suitable vanadyl complexes include, for example, vanadylacetylacetonate and vanadyl sulfate including vanadyl sulfate hydratessuch as vanadyl sulfate mono- and trihydrates.

Representative examples of tungsten and molybdenum complexes alsoinclude oxo complexes. Suitable oxo tungsten complexes include tungstateand tungsten oxide complexes. Suitable tungstate complexes includeammonium tungstate, calcium tungstate, sodium tungstate dihydrate, andtungstic acid. Suitable tungsten oxides include tungsten (IV) oxide andtungsten (VI) oxide. Suitable oxo molybdenum complexes includemolybdate, molybdenum oxide, and molybdenyl complexes. Suitablemolybdate complexes include ammonium molybdate and its hydrates, sodiummolybdate and its hydrates, and potassium molybdate and its hydrates.Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum(VI) oxide, and molybdic acid. Suitable molybdenyl complexes include,for example, molybdenyl acetylacetonate. Other suitable tungsten andmolybdenum complexes include hydroxo derivatives derived from, forexample, glycerol, tartaric acid, and sugars.

A wide variety of other anti-angiogenic factors may also be utilizedwithin the context of the present invention. Representative examplesinclude platelet factor 4; protamine sulphate; sulphated chitinderivatives (prepared from queen crab shells), (Murata et al., CancerRes. 51:22-26, 1991); Sulphated Polysaccharide Peptidoglycan Complex(SP-PG) (the function of this compound may be enhanced by the presenceof steroids such as estrogen, and tamoxifen citrate); Staurosporine;modulators of matrix metabolism, including for example, proline analogs,cishydroxyproline, d,L-3,4-dehydroproline, Thiaproline,alpha,alpha-dipyridyl, aminopropionitrile fumarate;4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate; Mitoxantrone;Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al., J.Bio. Chem. 267:17321-17326, 1992); Chymostatin (Tomkinson et al.,Biochem J. 286:475-480, 1992); Cyclodextrin Tetradecasulfate;Eponemycin; Camptothecin; Fumagillin (Ingber et al., Nature 348:555-557,1990); Gold Sodium Thiomalate (“GST”; Matsubara and Ziff. J. Clin.Invest. 79:1440-1446, 1987); anticollagenase-serum; alpha2-antiplasmin(Holmes et al., J. Biol. Chem. 262(4):1659-1664, 1987); Bisantrene(National Cancer Institute); Lobenzarit disodium(N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or “CCA”;Takeuchi et al., Agents Actions 36:312-316, 1992); Thalidomide;Angostatic steroid; AGM-1470; carboxynaminolmidazole; andmetalloproteinase inhibitors such as BB94.

Diseases at the Cellular Level

Diseases associated with increased cell survival or the inhibition ofapoptosis that could be treated, prevented, and/or diagnosed bycompositions of the present invention, include cancers (such asfollicular lymphomas, carcinomas with p53 mutations, andhormone-dependent tumors, including, but not limited to colon cancer,cardiac tumors, pancreatic cancer, melanoma, retinoblastoma,glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomachcancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma,osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma,breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer);autoimmune diseases, disorders, and/or conditions (such as, multiplesclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliarycirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemiclupus erythematosus and immune-related glomerulonephritis and rheumatoidarthritis) and viral infections (such as papilloma viruses, hepatitisvirus, herpes viruses, pox viruses and adenoviruses), inflammation,graft v. host disease, acute graft rejection, and chronic graftrejection. In preferred embodiments, polynucleotides, polypeptides,agonists and/or antagonists of the present invention are used to inhibitgrowth, progression, and/or metasis of cancers, in particular thoselisted above.

Additional diseases or conditions associated with increased cellsurvival that could be treated, prevented, and/or diagnosed by acomposition of the present invention include, but are not limited to,progression, and/or metastases of malignancies and related diseases,disorders, and/or conditions such as leukemia (including acute leukemias(e.g., acute lymphocytic leukemia, acute myelocytic leukemia (includingmyeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia)) and chronic leukemias (e.g., chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemiavera, lymphomas (e.g., fungoides mycosis, Hodgkin's disease andnon-Hodgkin's disease), multiple myeloma, Waldenstrom'smacroglobulinemia, heavy chain disease, and solid tumors including, butnot limited to, sarcomas and carcinomas such as fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, andretinoblastoma.

Diseases associated with increased apoptosis that could be treated,prevented, and/or diagnosed by a composition of the present inventioninclude AIDS; neurodegenerative diseases, disorders, and/or conditions(such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateralsclerosis, Retinitis pigmentosa, Cerebellar degeneration and brain tumoror prior associated disease); autoimmune diseases, disorders, and/orconditions (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto'sthyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease,polymyositis, systemic lupus erythematosus and immune-relatedglomerulonephritis and rheumatoid arthritis) myelodysplastic syndromes(such as aplastic anemia), graft v. host disease, ischemic injury (suchas that caused by myocardial infarction, stroke and reperfusion injury),liver injury (e.g., hepatitis related liver injury, ischemia/reperfusioninjury, cholestosis (bile duct injury) and liver cancer); toxin-inducedliver disease (such as that caused by alcohol), septic shock, cachexiaand anorexia.

Wound Healing and Epithelial Cell Proliferation

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing a composition of the presentinvention, for therapeutic purposes, for example, to stimulateepithelial cell proliferation and basal keratinocytes for the purpose ofwound healing, and to stimulate hair follicle production and healing ofdermal wounds. Compositions of the present invention, may be clinicallyuseful in stimulating wound healing including surgical wounds,excisional wounds, deep wounds involving damage of the dermis andepidermis, eye tissue wounds, dental tissue wounds, oral cavity wounds,diabetic ulcers, dermal ulcers, cubitus ulcers, arterial ulcers, venousstasis ulcers, burns resulting from heat exposure or chemicals, andother abnormal wound healing conditions such as uremia, malnutrition,vitamin deficiencies and complications associated with systemictreatment with steroids, radiation therapy and antineoplastic drugs andantimetabolites. Compositions of the present invention, could be used topromote dermal reestablishment subsequent to dermal loss

Compositions of the present invention could be used to increase theadherence of skin grafts to a wound bed and to stimulatere-epithelialization from the wound bed. Other uses of the compositionsof the present invention as they relate to wound healing and epithelialcell proliferation are listed in U.S. Pat. No. 6,472,512, which isincorporated herein by reference in its entirety.

Neurological Diseases

Nervous system diseases, disorders, and/or conditions, which can betreated with compositions of the present invention, include, but are notlimited to, nervous system injuries, and diseases, disorders, and/orconditions which result in either a disconnection of axons, a diminutionor degeneration of neurons, or demyelination. Nervous system lesionswhich may be treated in a patient (including human and non-humanmammalian patients) Other uses of the compositions of the presentinvention as they relate to treatment of nervous system relatedconditions are listed in U.S. Pat. No. 6,472,512, which is incorporatedherein by reference in its entirety.

In a specific embodiment, polynucleotides or polypeptides, as well asagonists or antagonists of the present invention of the invention areused to treat, prevent, detect, and/or diagnose multiple sclerosis.

The compositions of the invention which are useful for treating,preventing, and/or diagnosing a nervous system disorder may be selectedby testing for biological activity in promoting the survival ordifferentiation of neurons. For example, and not by way of limitation,compositions of the invention which elicit any of the following effectsmay be useful according to the invention: (1) increased survival time ofneurons in culture; (2) increased sprouting of neurons in culture or invivo; (3) increased production of a neuron-associated molecule inculture or in vivo, e.g., choline acetyltransferase oracetylcholinesterase with respect to motor neurons; or (4) decreasedsymptoms of neuron dysfunction in vivo. Such effects may be measured byany method known in the art. In preferred, non-limiting embodiments,increased survival of neurons may routinely be measured using a methodset forth herein or otherwise known in the art, such as, for example,the method set forth in Arakawa et al. (J. Neurosci. 10:3507-3515(1990)); increased sprouting of neurons may be detected by methods knownin the art, such as, for example, the methods set forth in Pestronk etal. (Exp. Neurol. 70:65-82 (1980)) or Brown et al. (Ann. Rev. Neurosci.4:1742 (1981)); increased production of neuron-associated molecules maybe measured by bioassay, enzymatic assay, antibody binding, Northernblot assay, etc., using techniques known in the art and depending on themolecule to be measured; and motor neuron dysfunction may be measured byassessing the physical manifestation of motor neuron disorder, e.g.,weakness, motor neuron conduction velocity, or functional disability.

Infectious Disease

Compositions of the present invention can be used to treat, prevent,and/or diagnose infectious agents. For example, by increasing the immuneresponse, particularly increasing the proliferation and differentiationof B and/or T cells, infectious diseases may be treated, prevented,and/or diagnosed. The immune response may be increased by eitherenhancing an existing immune response, or by initiating a new immuneresponse. Alternatively, compositions of the present invention may alsodirectly inhibit the infectious agent, without necessarily eliciting animmune response.

Viruses are one example of an infectious agent that can cause disease orsymptoms that can be treated, prevented, and/or diagnosed by apolynucleotide or polypeptide and/or agonist or antagonist of thepresent invention. Examples of viruses, include, but are not limited tothe following DNA and RNA viruses and viral families: Arbovirus,Adenoviridae, Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae,Caliciviridae, Circoviridae, Coronaviridae, Dengue, EBV, HIV,Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (such as,Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus (e.g.,Paramyxoviridae, Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g.,Influenza A, Influenza B, and parainfluenza), Papiloma virus,Papovaviridae, Parvoviridae, Picornaviridae, Poxyiridae (such asSmallpox or Vaccinia), Reoviridae (e.g., Rotavirus), Retroviridae(HTLV-I, HTLV-II, Lentivirus), and Togaviridae (e.g., Rubivirus).Viruses falling within these families can cause a variety of diseases orsymptoms, including, but not limited to: arthritis, bronchiollitis,respiratory syncytiai virus, encephalitis, eye infections (e.g.,conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B.C, E, Chronic Active, Delta), Japanese B encephalitis, Junin,Chikungunya, Rift Valley fever, yellow fever, meningitis, opportunisticinfections (e.g., AIDS), pneumonia, Burkitt's Lymphoma, chickenpox,condyloma acuminatum, laryngeal papillomatosis, hemorrhagic fever,Measles, Mumps, Parainfluenza, Rabies, the common cold, Polio, leukemia,Rubella, sexually transmitted diseases, skin diseases (e.g., Kaposi's,warts, fungoides mycosis), and viremia. polynucleotides or polypeptides,or agonists or antagonists of the invention, can be used to treat,prevent, and/or diagnose any of these symptoms or diseases. In specificembodiments, compositions of the invention are used to treat:meningitis, Dengue, EBV, and/or hepatitis (e.g., hepatitis B). In anadditional specific embodiment polynucleotides, polypeptides, oragonists or antagonists of the invention are used to treat patientsnonresponsive to one or more other commercially available hepatitisvaccines. In a further specific embodiment compositions of the inventionare used to treat, prevent, and/or diagnose AIDS.

In a specific embodiment, compositions of the present invention can beused to treat, detect, prevent and/or diagnose diseases associated withHepatitis virus (e.g., chronic hepatitis C, chronic hepatitis B, chronicactive hepatitis, and/or hepatitis D); diseases associated withPapilloma virus (e.g., condyloma acuminatum, warts, and/or laryngealpapillomatosis)

Similarly, bacterial or fungal agents that can cause disease or symptomsand that can be treated, prevented, and/or diagnosed by a polynucleotideor polypeptide and/or agonist or antagonist of the present inventioninclude, but are not limited to, the following Gram-Negative andGram-positive bacteria, bacterial families and fungi: Actinomyces (e.g.,Norcardia), Acinetobacter, Cryptococcus neoformans, Aspergillus,Bacillaceae (e.g., Bacillus anthrasis), Bacteroides (e.g., Bacteroidesfragilis), Blastomycosis, Bordetella, Borrelia (e.g., Borreliaburgdorferi), Brucella, Candidia, Campylobacter, Chlamydia, Clostridium(e.g., Clostridium botulinum, Clostridium dificile, Clostridiumperfringens, Clostridium tetani), Coccidioides, Corynebacterium (e.g.,Corynebacterium diptheriae), Cryptococcus, Dermatocycoses, E. coli(e.g., Enterotoxigenic E. coli and Enterohemorrhagic E. coli),Enterobacter (e.g. Enterobacter aerogenes), Enterobacteriaceae(Klebsiella, Salmonella (e.g., Salmonella typhi, Salmonella enteritidis,Salmonella paratyphi), Serratia, Yersinia, Shigella), Erysipelothrix,Haemophilus (e.g., Haemophilus influenza type B), Helicobacter,legionella (e.g., Legionella pneumophila), Leptospira, Listeria (e.g.,Listeria monocytogenes), Mycoplasma, Mycobacterium (e.g., Mycobacteriumleprae and Mycobacterium tuberculosis), Vibrio (e.g., Vibrio cholerae),Neisseriaceae (e.g., Neisseria gonorrhea, Neisseria meningitidis),Pasteurellacea, Proteus, Pseudomonas (e.g., Psuedomonas aeruginosa),Rickettsiaceae, Spirochetes (e.g., Treponema spp., Leptospira spp.,Borrelia spp.) Shigella spp., Staphylococcus (e.g., Staphylococcusaureus), Meningiococcus, Pneumococcus and Streptococcus (e.g.,Streptococcus pneumoniae and Groups A, B, and C Streptococci), andUreaplasmas. These bacterial, parasitic, and fungal families can causediseases or symptoms, including, but not limited to:antibiotic-resistant infections, bacteremia, endocarditis, septicemia,eye infections (conjunctivitis) tuberculosis, uveitis, gingivitis,bacterial diarrhea, opportunistic infections (e.g., AIDS relatedinfections), paronychia, prosthesis-related infections, dental caries,Reiter's Disease, respiratory tract infections (e.g., Whooping Cough orEmpyema), sepsis, Lyme Disease, Cat-Scratch Disease, dysentery,paratyphoid fever, food poisoning, Legionella disease, chronic and acuteinflammation, erythema, yeast infections, typhoid, pneumonia, gonorrhea,meningitis (e.g., meningitis types A and B), chlamydia, syphilis,diphtheria, leprosy, burcellosis, peptic ulcers, anthrax, spontaneousabortion, birth defects, lung infections, ear infections, deafness,blindness, lethargy, malaise, vomiting, chronic diarrhea, Crohn'sdisease, colitis, vaginosis, sterility, pelvic inflammatory disease,candidiasis, paratuberculosis, tuberculosis, lupus, botulism, gangrene,tetanus, impetigo, Rheumatic Fever, Scarlet Fever, sexually transmitteddiseases, skin diseases (e.g., cellulitis, dermatocycoses), toxemia,urinary tract infections, wound infections or noscomial infections.Compositions of the invention, can be used to treat or detect any ofthese symptoms or diseases. In specific embodiments,

Moreover, parasitic agents causing disease or symptoms that can betreated, prevented, and/or diagnosed by a composition of the presentinvention include, but not limited to, the following families or class:Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis,Dourine, Ectoparasitic, Giardiasis, Helminthiasis, Leishmaniasis,theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas andSporozoans (e.g., Plasmodium virax, Plasmodium falciparium, Plasmodiummalariae and Plasmodium ovale). These parasites can cause a variety ofdiseases or symptoms, including, but not limited to: Scabies,Trombiculiasis, eye infections, intestinal disease. (e.g., dysentery,giardiasis), liver disease, lung disease, opportunistic infections(e.g., AIDS related), malaria, pregnancy complications, andtoxoplasmosis.

Preferably, treatment or prevention using a composition of the presentinvention could either be by administering an effective amount of apolypeptide to the patient, or by removing cells from the patient,supplying the cells with a composition of the present invention, andreturning the cells to the patient (ex vivo therapy).

Regeneration

Compositions of the present invention can be used to differentiate,proliferate, and attract cells, leading to the regeneration of tissues.(See, Science 276:59-87 (1997).) the regeneration of tissues could beused to repair, replace, or protect tissue damaged by congenitaldefects, trauma (wounds, burns, incisions, or ulcers), age, disease(e.g. osteoporosis, osteocarthritis, periodontal disease, liverfailure), surgery, including cosmetic plastic surgery, fibrosis,reperfusion injury, or systemic cytokine damage.

Tissues that could be regenerated using the present invention includeorgans (e.g., pancreas, liver, intestine, kidney, skin, endothelium),muscle (smooth, skeletal or cardiac), vasculature (including vascularand lymphatics), nervous, hematopoietic, and skeletal (bone, cartilage,tendon, and ligament) tissue. Preferably, regeneration occurs without ordecreased scarring. Regeneration also may include angiogenesis.

Moreover, Compositions of the present invention may increaseregeneration of tissues difficult to heal. For example, increasedtendon/ligament regeneration would quicken recovery time after damage.Compositions of the present invention of the present invention couldalso be used prophylactically in an effort to avoid damage. Specificdiseases that could be treated, prevented, and/or diagnosed include oftendinitis, carpal tunnel syndrome, and other tendon or ligamentdefects. A further example of tissue regeneration of non-healing woundsincludes pressure ulcers, ulcers associated with vascular insufficiency,surgical, and traumatic wounds.

Similarly, nerve and brain tissue could also be regenerated by usingCompositions of the present invention to proliferate and differentiatenerve cells. Diseases that could be treated, prevented, and/or diagnosedusing this method include central and peripheral nervous systemdiseases, neuropathies, or mechanical and traumatic diseases, disorders,and/or conditions (e.g., spinal cord disorders, head trauma,cerebrovascular disease, and stoke). Specifically, diseases associatedwith peripheral nerve injuries, peripheral neuropathy (e.g., resultingfrom chemotherapy or other medical therapies), localized neuropathies,and central nervous system diseases (e.g., Alzheimer's disease,Parkinson's disease, Huntington's disease, amyotrophic lateralsclerosis, and Shy-Drager syndrome), could all be treated, prevented,and/or diagnosed using Compositions of the present invention.

Chemotaxis

Compositions of the present invention may have chemotaxis activity. Achemotaxic molecule attracts or mobilizes cells (e.g., monocytes,fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelialand/or endothelial cells) to a particular site in the body, such asinflammation, infection, or site of hyperproliferation. The mobilizedcells can then fight off and/or heal the particular trauma orabnormality.

Compositions of the present invention may increase chemotaxic activityof particular cells. These chemotactic molecules can then be used totreat, prevent, and/or diagnose inflammation, infection,hyperproliferative diseases, disorders, and/or conditions, or any immunesystem disorder by increasing the number of cells targeted to aparticular location in the body. For example, chemotaxic molecules canbe used to treat, prevent, and/or diagnose wounds and other trauma totissues by attracting immune cells to the injured location. Chemotacticmolecules of the present invention can also attract fibroblasts, whichcan be used to treat, prevent, and/or diagnose wounds.

It is also contemplated that Compositions of the present invention mayinhibit chemotactic activity. These molecules could also be used totreat, prevent, and/or diagnose diseases, disorders, and/or conditions.Thus, Compositions of the present invention could be used as aninhibitor of chemotaxis.

Formulations

The compositions of the present invention may be formulated and dosed inany effective manner consistent with good medical practice, taking intoaccount the clinical condition of the individual patient (especially theside effects of treatment with the polypeptide alone), the site ofdelivery of the polypeptide composition, the method of administration,the scheduling of administration, and other factors known topractitioners. The “effective amount” of polypeptide to be administeredmay be determined by such considerations.

One embodiment of the present invention provides for pharmaceuticalcompositions comprising a polypeptide of the present invention. Theinvention also provides for pharmaceutical compositions comprisingcombinations of polypeptides of the present invention. In oneembodiment, the invention provides for a pharmaceutical compositionscomprising a combination of polypeptides of the present invention,wherein the ratio of IFN-λ1:IFN-λ2:IFN-λ3 is described by the formulam:n:o, wherein m may be any number between 0 and 1000, n may be anynumber between 0 and 1000 and o may be any number between 0 and 1000,and wherein at least one of m, n or o is different from 0. The inventionalso provides for pharmaceutical compositions comprising a combinationof polypeptides of the present invention, wherein the ratio ofIFN-λ1:IFN-λ2:IFN-λ3 is described by the formula m:n:o, wherein m may beany number between 0 and 1000, n may be any number between 0 and 1000and o may be any number between 0 and 1000, and wherein at least two ofm, n or o are different from 0. The invention further provides forpharmaceutical compositions comprising a combination of polypeptides ofthe present invention, wherein the ratio of IFN-λ1:IFN-λ2:IFN-λ3 isdescribed by the formula m:n:o, wherein m may be any number between 0and 1000, n may be any number between 0 and 1000 and o may be any numberbetween 0 and 1000, and wherein all three of m, n and o are differentfrom 0. The combination may further comprise antibodies of the presentinvention. The combination may also further comprise an agonist and/orantagonist, or a combination of agonists and/or antagonists of theCRF2-12 receptor the CRF2-12 receptor complex. In a preferredembodiment, the combination further comprises an antibody to theextracellular domain of CRF2-12 that acts as an agonist of the CRF2-12receptor or the CRF2-12 receptor complex. The combination may furthercomprise other cytokines, interleukins and/or other pharmaceuticals,including anti-inflamatory agents.

The invention also provides for pharmaceutical compositions comprisingan antibody of the present invention. The invention also provides forpharmaceutical compositions comprising combinations of antibodies of thepresent invention. In one embodiment, the invention provides acomposition comprising a combination of anti-IFN-λ1 specific antibody,anti-IFN-λ2 specific antibody and anti-IFN-λ3 specific antibody, whereinthe ratio of anti-IFN-λ1 specific antibody: anti-IFN-λ2 specificantibody: anti-IFN-λ3 specific antibody is described by the formulax:y:z, wherein x may be any number between 0 and 1000, y may be anynumber between 0 and 1000 and z may be any number between 0 and 1000,and wherein at least one of x, y or z is different from 0. The inventionalso provides for pharmaceutical compositions comprising a combinationof anti-IFN-λ1 specific antibody, anti-IFN-λ2 specific antibody andanti-IFN-λ3 specific antibody, wherein the ratio of anti-IFN-λ1 specificantibody: anti-IFN-λ2 specific antibody: anti-IFN-λ3 specific antibodyis described by the formula x:y:z, wherein x may be any number between 0and 1000, y may be any number between 0 and 1000 and z may be any numberbetween 0 and 1000, and wherein at least two of x, y or z are differentfrom 0. The invention also provides for pharmaceutical compositioncomprising a combination of anti-IFN-λ1 specific antibody, anti-IFN-λ2specific antibody and anti-IFN-λ3 specific antibody, wherein the ratioof anti-IFN-λ1 specific antibody: anti-IFN-λ2 specific antibody:anti-IFN-λ3 specific antibody is described by the formula x:y:z, whereinx may be any number between 0 and 1000, y may be any number between 0and 1000 and z may be any number between 0 and 1000, and wherein allthree of x, y or z are different from 0. The combination may furthercomprise an IFN-λ or a combination of IFN-λs of the present invention.The combination may also further comprise an agonist and/or antagonist,or a combination of agonists and/or antagonists of the CRF2-12 receptorthe CRF2-12 receptor complex. In a preferred embodiment, the combinationfurther comprises an antibody to the extracellular domain of CRF2-12that acts as an agonist of the CRF2-12 receptor or the CRF2-12 receptorcomplex. The combination may further comprise other cytokines,interleukins and/or other pharmaceuticals, including anti-inflamatoryagents.

Any effective amount of the polypeptides or polypeptide fragments of thepresent invention may be administered to an individual in need thereof.As a general proposition, the total pharmaceutically effective amountadministered parenterally per dose will be in the range of about 1μg/kg/day to 10 mg/kg/day of patient body weight, although, as notedabove, this will be subject to therapeutic discretion. More preferably,this dose is at least 0.01 mg/kg/day, and most preferably for humansbetween about 0.01 and 1 mg/kg/day. If given continuously, thecomposition is typically administered at a dose rate of about 1μg/kg/hour to about 50 μg/kg/hour, either by 14 injections per day or bycontinuous subcutaneous infusions, for example, using a mini-pump. Anintravenous bag solution may also be employed. The length of treatmentneeded to observe changes and the interval following treatment forresponses to occur appears to vary depending on the desired effect.

Pharmaceutical compositions containing the polypeptides or polypeptidefragments of the invention may be administered by any effective route,including, for example, orally, rectally, parenterally, intracistemally,intravaginally, intraperitoneally, topically (as by powders, ointments,drops or transdermal patch), bucally, or as an oral or nasal spray.

By “pharmaceutically acceptable carrier” is meant any effectivepharmaceutically acceptable carriera, preferably a non-toxic solid,semisolid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type.

The term “parenteral” as used herein refers to any effective parenteralmode of administration, including modes of administration such asintravenous, intramuscular, intraperitoneal, intrasternal, subcutaneousand intraarticular injection and infusion.

The compositions may also suitably be administered by sustained-releasesystems. Suitable examples of sustained-release compositions includesemi-permeable polymer matrices in the form of shaped articles, e.g.,films, or microcapsules. Sustained-release matrices include polylactides(U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman, U. et al., Biopolymers 22:547-556(1983)), poly (2-hydroxyethyl methacrylate) (R. Langer et al., J.Biomed. Mater. Res. 15:167-277 (1981), and R. Langer, Chem. Tech.12:98-105 (1982)), ethylene vinyl acetate (R. Langer et al., Id.) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988).

In a preferred embodiment, compositions of the invention are formulatedin a biodegradable, polymeric drug delivery system, for example asdescribed in U.S. Pat. Nos. 4,938,763; 5,278,201; 5,278,202; 5,324,519;5,340,849; and 5,487,897 and in International Publication NumbersWO01/35929, WO00/24374, and WO00/06117 which are hereby incorporated byreference in their entirety. In specific preferred embodiments thecompositions of the invention are formulated using the ATRIGELBiodegradable System of Atrix Laboratories, Inc. (Fort Collins, Colo.).

Examples of biodegradable polymers which can be used in the formulationof compositions of the present invention, include but are not limitedto, polylactides, polyglycolides, polycaprolactones, polyanhydrides,polyamides, polyurethanes, polyesteramides, polyorthoesters,polydioxanones, polyacetals, polyketals, polycarbonates,polyorthocarbonates, polyphosphazenes, polyhydroxybutyrates,polyhydroxyvalerates, polyalkylene oxalates, polyalkylene succinates,poly(malic acid), poly(amino acids), poly(methyl vinyl ether),poly(maleic anhydride), polyvinylpyrrolidone, polyethylene glycol,polyhydroxycellulose, chitin, chitosan, and copolymers, terpolymers, orcombinations or mixtures of the above materials. The preferred polymersare those that have a lower degree of crystallization and are morehydrophobic. These polymers and copolymers are more soluble in thebiocompatible solvents than the highly crystalline polymers such aspolyglycolide and chitin which also have a high degree ofhydrogen-bonding. Preferred materials with the desired solubilityparameters are the polylactides, polycaprolactones, and copolymers ofthese with glycolide in which there are more amorphous regions toenhance solubility. In specific preferred embodiments, the biodegradablepolymers which can be used in the formulation of compositions arepoly(lactide-co-glycolides).

Polymer properties such as molecular weight, hydrophobicity, andlactide/glycolide ratio may be modified to obtain the desired drugrelease profile (See, e.g., Ravivarapu et al., Journal of PharmaceuticalSciences 89:732-741 (2000), which is hereby incorporated by reference inits entirety).

It is also preferred that the solvent for the biodegradable polymer benon-toxic, water miscible, and otherwise biocompatible. Examples of suchsolvents include, but are not limited to, N-methyl-2-pyrrolidone,2-pyrrolidone, C2 to C6 alkanols, C1 to C15 alcohols, dils, triols, andtetraols such as ethanol, glycerine propylene glycol, butanol; C3 to C15alkyl ketones such as acetone, diethyl ketone and methyl ethyl ketone;C3 to C15 esters such as methyl acetate, ethyl acetate, ethyl lactate;alkyl ketones such as methyl ethyl ketone, C1 to C15 amides such asdimethylformamide, dimethylacetamide and caprolactam; C3 to C20 etherssuch as tetrahydrofuran, or solketal; tweens, triacetin, propylenecarbonate, decylmethylsulfoxide, dimethyl sulfoxide, oleic acid,1-dodecylazacycloheptan-2-one, Other preferred solvents are benzylalchohol, benzyl benzoate, dipropylene glycol, tributyrin, ethyl oleate,glycerin, glycofural, isopropyl myristate, isopropyl palmitate, oleicacid, polyethylene glycol, propylene carbonate, and triethyl citrate.The most preferred solvents are N-methyl-2-pyrrolidone, 2-pyrrolidone,dimethyl sulfoxide, triacetin, and propylene carbonate because of thesolvating ability and their compatibility.

Additionally, formulations comprising compositions of the presentinvention and a biodegradable polymer may also include release-ratemodification agents and/or pore-forming agents. Examples of release-ratemodification agents include, but are not limited to, fatty acids,triglycerides, other like hydrophobic compounds, organic solvents,plasticizing compounds and hydrophilic compounds. Suitable release ratemodification agents include, for example, esters of mono-, di-, andtricarboxylic acids, such as 2-ethoxyethyl acetate, methyl acetate,ethyl acetate, diethyl phthalate, dimethyl phthalate, dibutyl phthalate,dimethyl adipate, dimethyl succinate, dimethyl oxalate, dimethylcitrate, triethyl citrate, acetyl tributyl citrate, acetyl triethylcitrate, glycerol triacetate, di(n-butyl)sebecate, and the like;polyhydroxy alcohols, such as propylene glycol, polyethylene glycol,glycerin, sorbitol, and the like; fatty acids; triesters of glycerol,such as triglycerides, epoxidized soybean oil, and other epoxidizedvegetable oils; sterols, such as cholesterol; alcohols, such as C6-C12alkanols, 2-ethoxyethanol. The release rate modification agent may beused singly or in combination with other such agents. Suitablecombinations of release rate modification agents include, but are notlimited to, glycerin/propylene glycol, sorbitol/glycerine, ethyleneoxide/propylene oxide, butylene glycol/adipic acid, and the like.Preferred release rate modification agents include, but are not limitedto, dimethyl citrate, triethyl citrate, ethyl heptanoate, glycerin, andhexanediol. Suitable pore-forming agents that may be used in the polymercomposition include, but are not limited to, sugars such as sucrose anddextrose, salts such as sodium chloride and sodium carbonate, polymerssuch as hydroxylpropylcellulose, carboxymethylcellulose, polyethyleneglycol, and polyvinylpyrrolidone. Solid crystals that will provide adefined pore size, such as salt or sugar, are preferred.

In specific preferred embodiments the compositions of the invention areformulated using the BEMA BioErodible Mucoadhesive System, MCAMucoCutaneous Absorption System, SMP Solvent MicroParticle System, orBCP BioCompatible Polymer System of Atrix Laboratories, Inc. (FortCollins, Colo.).

Sustained-release compositions also include liposomally entrappedpolypeptides. Liposomes containing a polypeptide of the presentinvention are prepared by methods known, for example: DE 3,218,121;Epstein et al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwanget al., Proc. Natl. Acad. Sci. (USA) 77:4030-4034 (1980); EP 52,322; EP36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl.83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324.Ordinarily, the liposomes are of the small (about 200-800 Angstroms)unilamellar type in which the lipid content is greater than about 30mol. percent cholesterol, the selected proportion being adjusted foreffective polypeptide therapy.

A liposomally entrapped polypeptide of the present invention can beproduced for clinically effective formulations. Modified interferons,such as PEG-Intron (Schering-Plough INTRON) are clinically effective andcan be used in therapies with other agents, such as Robavirin.

The polypeptide of the present invention may be administered incombination with other known anti-viral, immunomodulatory andanti-proliferative therapies, such as IL-2, alpha interferon, KDI,Ribavirin and temozolomide.

For parenteral administration, in one embodiment, the polypeptide of theinvention is formulated generally by mixing it at the desired degree ofpurity, in a unit dosage injectable form (solution, suspension, oremulsion), with a pharmaceutically acceptable carrier, i.e., one that isnon-toxic to recipients at the dosages and concentrations employed andis compatible with other ingredients of the formulation. For example,the formulation preferably does not include oxidizing agents and othercompounds that are known to be deleterious to polypeptides.

Generally, the formulations are prepared by contacting the polypeptideuniformly and intimately with liquid carriers or finely divided solidcarriers or both. Then, if necessary, the product is shaped into thedesired formulation. Preferably the carrier is a parenteral carrier,more preferably a solution that is isotonic with the blood of therecipient. Examples of such carrier vehicles include water, saline,Ringer's solution, and dextrose solution. Non-aqueous vehicles such asfixed oils and ethyl oleate are also useful herein, as well asliposomes.

The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, manose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

The polypeptide is typically formulated in such vehicles at aconcentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, ata pH of about 3 to 8. It will be understood that the use of certain ofthe foregoing excipients, carriers, or stabilizers may result in theformation of polypeptide salts.

Polypeptide to be used for therapeutic administration must be sterile.Sterility may be achieved in any effective manner. For example, byfiltration through sterile filtration membranes (e.g., 0.2 micronmembranes). Therapeutic compositions generally are placed into acontainer having a sterile access port, for example, an intravenoussolution bag or vial having a stopper pierceable by a hypodermicinjection needle.

Compositions of the present invention ordinarily will be stored in unitor multi-dose containers, for example, sealed ampoules or vials, as anaqueous solution or as a lyophilized formulation for reconstitution. Asan example of a lyophilized formulation, 10-ml vials are filled with 5ml of sterile-filtered 1% (w/v) aqueous polypeptide solution, and theresulting mixture is lyophilized. The infusion solution is prepared byreconstituting the lyophilized polypeptide using bacteriostaticWater-for-Injection.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, thepolypeptides of the present invention may be employed in conjunctionwith other therapeutic compounds.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

EXAMPLES Example 1 Cloning of the CRF2-12 cDNA Expression Pattern andProtein Characterization

A search of the GenBank database was performed with the TBLASTN programusing as a query a consensus amino acid motif shared by extracellulardomains of class II cytokine receptor family members. A genomic fragmentfrom human chromosome I encoding a novel amino acid (a.a.) sequence withhomology to the consensus motif was identified. This genomic fragmentmapped in the vicinity of a known member of the family, namely, theIL-22R1 (CRF2-9) gene (1p36+11 chromosomal region). According to thepresent invention this fragment represented an exon of a novel receptorfrom this family of receptors.

The existence of a gene (Hs1_(—)4548_(—)30_(—)10_(—)1) in this regionwas predicted by GenomeScan (NCBI web site) (5). However, according tothe present invention the last four exons were predicted incorrectly bythe program, and the last exon encoding intracellular domain of thereceptor of the present invention is different and distinct from thatprovided by GenomeScan.

Several ESTs were positioned in this genomic region representing the 3′untranslated region (3′-UTR) of a novel gene (the UniGene designationHs.105866), which we predicted to be contingent with theHs1_(—)4548_(—)30_(—)10_(—)1 gene.

In accordance with the present invention, several sets of primers weredesigned and used for RT-PCR with several human cDNA libraries as atemplate to obtain the receptor of the present invention. The primersand a human placental cDNA library (Clontech, catalog #HL4025AH) wereused to obtain the genomic organization and exon-intron junctions of theCRF2-12 gene.

Primers

5′-CCAGAACTTCAGCGTGTACCTGAC-3′ (R12-10 forward primer, R12-10F),5′-CCAGGATGTGACCTATTTTGTGGC-3′ (R12-11F),5′-CCAGCAAGAAGCAGGTGGGCTTAG-3 ′ (R12-13 reverse primer, R12-13R),5′-TTCGGGACACTGAACGTGTAGATG-3′ (R12-14R).

Libraries containing cDNA isolated from HeLa cells (Clontech, catalog#HL4048AH) or from various human tissues, including placenta, fetalbrain and liver, EBV-transformed lymphocytes, and leukocytes (Clontech,catalog #HL4025AH, HL4028AH, HL4029AH, HL4006AE and HL4050AH) were usedfor nested PCR to assess the presence of CRF2-12 cDNA in theselibraries.

A first round was performed with R12-10F and R12-13R primers followed bythe second round with R12-11F and R12-14R primers and the PCR product ofthe first round as a template.

The placental cDNA library was selected and further used foramplification of CRF2-12 cDNA by nested PCR. First round was performedwith primers:

5′-GCCGCGGCAGGAAGGCCATGGCG-3′ (R12-1F) and5′-CATCCTCCTCCTCTTCGTCCTCTG-3′ (R12-16R).

Then the PCR product of the first round was used as a template for thesecond round of PCR amplification with either primers.

5′-GCCGGTACCATGGCGGGGCCCGAGCGCTGG-3′ (R12-2F) and5′-AGGGCTAGCCAGTTGGCTTCTGGGACCTCC-3′ (R12-9R) or5′-GCCGGATCCCCGTCTGGCCCCTCCCCAGAA-3′ (R12-3F) and5′-AGGGCTAGCCAGTTGGCTTCTGGGACCTCC-3′ (R12-9R)

to clone the extracellular domain of the CRF2-12 protein into eitherplasmid pEF3-IL-10R1/γR1 (Kotenko, et al., (1997) EMBO J. 16, 5894-5903)with the use of KpnI and NheI restriction endonucleases, resulting inplasmid pEF-CRF2-12/γR1, or plasmid pEF3-FLγR2/αR2c (Kotenko, et al.,(1999) Proc Natl Acad Sci USA 96, 5007-5012) with the use of BamHI andNheI restriction endonucleases, resulting in plasmidpEF-FL-CRF2-12/αR2c.

The intracellular domain of IFN-αR2c was replaced by the intracellulardomain of IFN-γR1 by cloning of the fragment containing theintracellular domain of IFN-γR1 from plasmid pEF-CRF2-12/R1 into plasmidpEF-FL-CRF2-12/αR2c with the use of NheI and BssHII restrictionendonucleases, resulting in plasmid pEF-FL-CRF2-12/γR1.

Cloning of the CRF2-12 intracellular domain.

Primers

5′-TCTAAGCCCACCTGCTTCTTGCTG-3′ (R12-12F),5′-CTGGCTAGCCCTGGTGCTGCCATCGC-3′ (R12-19F),5′-AGGCAGCAGCAGCATCAGATTCGG-3′ (R12-8R) and5′-GGGTCTAGATCACCTGGCCATGTAATGCCCCA-3′ (R12-7R).

Nested PCR of the same placental cDNA library with the above primers(R12-12F and R12-8R primers for the first round and R12-19F and R12-7Rprimers for the second round) to clone the CRF2-12 intracellular domaininto the pCR2.1 vector (Invitrogen).

The CRF2-12 intracellular domain was then cloned into plasmidspEF-CRF2-12/γR1 and pEF-FL-CRF2-12/γR1 with the use of NheI and NotIrestriction endonucleases, resulting in plasmids pEF-CRF2-12 andpEF-FLCRF2-12, respectively.

Plasmid pEF-CRF (pEF-CRF2-4 or pEF-IL-10R2) was previously described(Kotenko, et al., (1997) EMBO J. 16, 5894-5903). Plasmid pEF-CRF2-11 (orpEF2-IL-20R2) was obtained by cloning PCR-derived CRF2-11 cDNA fragmentinto the pcDEF3 vector (Goldman 1996) with the use of KpnI and EcoRIrestriction endonucleases.

Accordingly, a human cDNA of the receptor of the present invention wasobtained having SEQ. ID. NO. 1 and encoding a receptor having an aminoacid sequence in SEQ. ID. NO. 2. The novel receptor of the presentinvention was designated CRF2-12 (12th member of the class II cytokinereceptor family; FIG. 14A).

Example 2 CRF2-12 Characterization

In accordance with the present invention, CRF2-12 was found to possess acharacteristic signal peptide of about 18 a.a. and a 23 a.a.transmembrane domain (227 to 249 a.a.), which divides the receptor intoa 208 a.a. extracellular domain and a relatively long 271a.a.intracellular domain (FIG. 14A).

Alignment of the extracellular domains of the class II cytokinereceptors assigned CRF2-12 in a subgroup with several other receptors,CRF2-8, CRF2-9, CRF2-10, IL-10R1 and IFN-γR (FIG. 14B).

In accordance with the present invention, the entire CRF2-12 gene of theinvention is composed of 7 exons (FIG. 14C). The structure of the geneof the present invention was found to correlate with the commonconserved architecture of other genes encoding CRF members (Kotenko2002).

The first exon of the CRF2-12 gene of the present invention contains the5′-UTR and the signal peptide. Exons 2, 3, 4 and 5 and a part of exon 6of the CRF2-12 gene of the present invention encode the extracellulardomain. Exon 6 of the CRF2-12 gene of the present invention also encodesthe transmembrane domain and the beginning of the intracellular domain.Exon 7 of the CRF2-12 gene of the present invention encodes the rest ofthe intracellular domain and the 3′-UTR.

Both genes, the CRF2-12 gene and the IL-22R1 gene, are transcribed inthe same direction toward the telomere and positioned approximately 10kb apart. All exon/intron and intron/exon splice sites within of theCRF2-12 gene of the present invention conform well to the consensusmotifs (exon/GT-intron-AG/exon).

Example 3 CRF2-12 Expression

To determine what cell types express CRF2-12, a panel of RNA derivedfrom various tumor cell lines was evaluated.

Expression of CRF2-12 mRNA.

Northern blotting was performed as described (Gallagher, et al., (2000)Genes Immun 1, 442-450 and Kotenko, et al., (2001) J Biol Chem 276,2725-2732) with the use of a CRF2-12 probe corresponding to the codingregion of the CRF2-12 cDNA.

Northern blotting was performed on two blots containing mRNA isolatedfrom: human cancer cell lines (promyelocytic leukemia HL-60, epitheloidcarcinoma HeLa S3, lymphoblastic leukemia MOLT-4, Burkitt's lymphomaRaji, colorectal adenocarcinoma, SW480, lung carcinoma A549 and melanomaG361) and normal human fetal tissues (heart, kidney, skin, smallintestine) and adult lung (FIGS. 15A and 15B). Arrows point to theCRF2-12 and the b-actin transcripts. Equal RNA loading was assessed byevaluating the expression of the b-actin gene.

CRF2-12 mRNA was expressed at variable levels in all of the cell linesexamined (FIG. 15A). These included both hematopoietic (HL-60, K-562,MOLT4 and Raji) and non-hematopoietic (HeLa, SW480, A549, and G-361)cell lines. Of these cell lines, Raji cells displayed the highest levelsof CRF2-12 gene expression. CRF2-12 was also expressed by various normaltissues, including heart, kidney, skin, small intestine, and lung (F1G.15B). CRF2-12 mRNA was also present in skeletal muscle and liver.Therefore, CRF2-12 appears to be constitutively expressed in a varietyof cell lines and tissues.

Example 4 Construction of Tandem Vectors Encoding Two Receptors andConstruction of a Chimeric CRF2-12/IFN-γR1 Receptor

To assay for the CRF2-12 receptor ligands, a chimeric CRF2-12/IFN-γR1(CRF2-12/γR1) receptor (FIG. 20A) was created. The chimericCRF2-12/IFN-γR1 receptor was expressed with different intact R2 chainsin various combinations (CRF2-4 and CRF2-11) in 16-9 hamster cells.

To ensure that both receptors were expressed in a single transfectedcell, tandem vectors, in which expression of CRF2-12/γR1 and eitherCRF2-4 or CRF2-11 is controlled by separate promoters andpolyadenylation signal, were constructed. Tandem vectors encoding tworeceptors, CRF2-12/γR1 and either CRF24 or CRF2-11, in which theexpression of each receptor is controlled by separate promoters andpolyadenylation signals were created as follows. The fragment containingthe EF-1α promoter, the CRF2-12 coding sequence, and the bovine growthhormone (BGH) polyadenylation signal was released from thepEF-CRF2-12/γR1 vector by digestion with BsaI and BssHII restrictionendonucleases and ligated into the BsaI and MluI sites of either thepEF-CRF24 or pEF-CRF2-11 plasmid. The resulting plasmids were designatedpEF-CRF2-12/γR1+CRF2-4 and pEF-CRF2-12/γR1+CRF2-11, respectively.

Example 6 Testing Known Cytokines for CRF2-12 Specificity

Plasmids pEF-CRF2-12/γR1+CRF24 and pEF-CRF2-12/γR1+CRF2-11 weretransfected into hamster cells and cellular pools of G418-resistantclones were collected. The ability of different cytokines to activatethe chimeric receptor and the ability of resultant receptor complexes totransduce IFN-γ-like signaling in the transfected hamster cells wastested. In particular, the chimeric receptor was tested forresponsiveness to different ligands utilizing EMSA to detect Stat1activation and FACS to evaluate MHC class I antigen expression, theactivities characteristic for IFN-γ signaling. Signaling by IL-1 g,IL-20, IL-22 and IL-24 in cells expressing CRF2-12/γR1 and either CRF24or CRF2-11 chains was not observed.

Example 7 CRF2-12 Ligand Identification and Cloning

In accordance with the present invention, a group of highly conservedbetween themselves proteins named IFN-λ1, IFN-λ2 and IFN-λ3 which alsodemonstrated very limited primary homology to members of both type I IFNand IL-10 families, were identified. (FIGS. 16, 17, 18 and 19, SEQ. IDNO. 7, SEQ. ID NO. 8, SEQ. ID NO. 9, SEQ. ID NO. 10, SEQ. ID NO. 11,SEQ. ID NO. 12).

Genes for these three novel cytokines were cloned as follows.

Human genomic DNA and the following primers were used to amplify IFN-λgenes:

5′-GCGGTACCATGGCTGCAGCTTGGACCGTGG-3′, (ifnl-1F)5′-GCGGTACCATGACTGGGGACTGCACGCCAGTG-3′, (ifnl-2F)5′-GCGGATCCTGTCCCCACTTCCAAGCCCACC-3′, (ifnl-5F)5′-GCGGATCCTGTCGCCAGGCTCCGCGGGGCT-3′ (ifnl-6F)5′-CGGAATTCAGGTGGACTCAGGGTGGGTTGA-3′ (ifnl-3R) and5′-CGGAATTCAGACACACAGGTCCCCACTGGCAACACA-3′. (ifnl-4R)

First round was performed with either ifnl-1F and ifnl-3R primers orifnl-2F and ifnl-1-4R primers followed by the second round with eitherifnl-5F and ifnl-3R primers or ifnl-6F and ifnl-4R primers andcorresponding PCR products of the first round as a template. ResultingPCR products were cloned into the pEF-SPFL vector with the use of BamHIand EcoRI restriction endonucleases, generating plasmid pEF-FL-IFN-λ1gene, pEF-FL-IFN-λ2gene and pEF-FL-IFN-λ3gene.

The PCR-derived genomic fragments which encode mature IFN-λ1, IFN-λ2,IFN-λ3 proteins (Pro23 (FIG. 19A) was predicted to be the first a.a. ofthe mature proteins) were cloned into plasmid pEF-SPFL, resulting inplasmids pEF-FL-IFN-λ1gene, pEF-FL-IFN-λ2gene and pEF-FL-IFN-λ3gene.

Plasmid pEF-SPFL encodes the signal peptide derived from the humanIFN-γR2 chain followed by the FLAG epitope. The pEF-FL-IFN-λ1 gene,pEF-FL-IFN-λ2gene and pEF-FL-IFN-λ3gene constructs abutted each ofIFN-λs reading frames to the frame of the FLAG epitope (FL-IFN-λs).

IFN-λs cDNAs were subsequently cloned by RT-PCR using as a templatemRNAs from COS-1 cells independently transfected with plasmidspEF-FL-IFN-λ1gene, pEF-FL-IFN-λ2gene and pEF-FL-IFN-λ3gene. cDNAfragments encoding mature IFN-λs proteins were cloned into the sameplasmid pEF-SPFL resulting in plasmids pEF-FL-IFN-λ1, pEF-FL-IFN-λ2 andpEF-FL-IFN-λ3.

The same primers as for cloning genomic fragments and total RNA isolatedfrom COS-1 cells transfected with plasmids pEF-FL-IFN-λ1 gene,pEF-FL-IFN-λ2gene and pEF-FL-IFN-λ3gene were used for RT-PCR to obtainIFN-λ1, IFN-λ2 and IFN-λ3 cDNA fragments, which were subsequently clonedinto plasmid pEF-SPFL with the use of BamHI and EcoRI restrictionendonucleases, resulting in plasmids pEF-FL-IFN-λ1, pEF-FL-IFN-λ2 andpEF-FL-IFN-λ3. These plasmids encode IFN-λ molecules tagged at theirN-terminus with the FLAG epitope (FL-IFN-λ).

The PCR product obtained with primers:

ifnl-5F and

5′-GCGAATTCATGCGACGGATGCCCGCCGAATTGAGGTGGACTCAGGGT GGG-3′ (ifn1-7R).

and plasmid pEF-FL-IFN-λ1 as a template was digested with BamHI andEcoRI restriction endonucleases and cloned into corresponding sites ofthe pEF-SPFL vector, resulting in plasmid pEF-FL-IFN-λ1-P. This plasmidencodes FL-IFN-λ tagged at its C-terminus with theArg-Arg-Ala-Ser-Val-Ala sequence (FL-IFN-λ-P), that contains theconsensus amino acid sequence recognizable by the catalytic subunit ofthe cAMP-dependent protein kinase (Li, et al., (1989) Proc Natl Acad SciUSA 86, 558-562).

The PCR product obtained with primers 5′-GATAGTACTTCCAAGCCCACCACA-3′(ifnl-5F) and ifnl-3R and plasmid pEF-FL-IFN-λ1 as a template wasdigested with ScaI and EcoRI restriction endonucleases and cloned intoXmnI and EcoRI sites of the pMAL-p2x vector (New England Biolabs),resulting in plasmid pMAL-IFN-λ1. This plasmid encodes MBP(maltose-binding protein)-IFN-λ fusion protein which is secreted to theperiplasm of E. coli cells. The fusion protein was purified from theperiplasm by the cold osmotic shock method according to themanufacturer's protocol.

In accordance with the present invention, IFN-λ1, IFN-λ2, IFN-λ3 werefound to be encoded by genes including five exons, which resemble thestructural organization of genes encoding IL-10-related cytokines.

Genes encoding three members of this family, designated IFN-λ1, IFN-λ2and IFN-λ3, (FIGS. 16, 17, 18 and 19, SEQ. ID NO. 7, SEQ. ID NO. 8, SEQ.ID NO. 9, SEQ. ID NO. 10, SEQ. ID NO. 11, SEQ. ID NO. 12) were found tobe clustered on human chromosome 19 (19q13+13 region).

Example 8

Expression of IFN-λs from pEF-FL-IFN-λ1 gene, pEF-FL-IFN-λ2gene andpEF-FL-IFN-λ3gene.

COS-1 cells were transiently transfected with pEF-FL-IFN-λ1 gene,pEF-FL-IFN-λ2gene and pEF-FL-IFN-α3gene plasmids and three days aftertransfection conditioned media was collected and subjected to Westernblotting and biological assays (FIGS. 20, 21, 22, 23 and 24). Westernblotting revealed that FL-IFN-λs were secreted from COS cells andmigrated on the SDS-PAGE gel as several bands in the region of about 35kDa for IFN-λ1 and as a single band of about 25 kDa for IFN-λ2 andIFN-λ3 proteins (FIG. 20C). In accordance with the present invention,this observation suggests glycosylation of IFN-λ1. Further in accordancewith the present invention, a potential site for N-linked glycosylation(Asn-X-Thr/Ser) is present in IFN-λ1 (FIG. 19A).

COS cells conditioned media containing IFN-λs were used to treat hamstercells expressing CRF2-12/γR1 and either CRF2-4 or CRF2-11 chains. Allthree proteins were able to induce Stat activation and up-regulate MHCclass I antigen expression, but only in cells expressing bothCRF2-12/γR1 and CRF2-4 chains (FIG. 21A, 23). Expression of neitherCRF2-12/γR1 nor CRF2-4 alone rendered cells responsive to IFN-λtreatment.

Three days after transfection, conditioned media from COS-1 cellstransiently transfected with expression plasmids were collected.FLAG-tagged proteins in the conditioned media were identified by Westernblotting with anti-FLAG epitope specific M2 monoclonal antibody (Sigma)as described (Kotenko, et al., (2000) Proc Natl Acad Sci USA 97,1695-1700).

Conditioned media from COS-1 cells independently transiently transfectedwith plasmids pEF-FL-IFN-λ1, pEF-FL-IFN-λ2 and pEF-FL-IFN-λ3demonstrated indistinguishable biological activities and Westernblotting pattern as conditioned media from COS-1 cells independentlytransfected with plasmids pEF-FL-IFN-λ1gene, pEF-FL-IFN-λ2gene andpEF-FL-IFN-λ3gene (FIGS. 20C, 21A, 21B and 23).

In accordance with another embodiment of the present invention, the useof flow cytometry was used to detect binding of FLAG epitope-taggedIFN-λs to the cells expressing both CRF2-12/γR1 and CRF24 chains (FIG.21A).

In accordance with another embodiment of the present invention,FL-IFN-λs were found to compete for receptor binding (FIG. 22). Cellsexpressing either chain alone did not bind FL-IFN-λs as measured byFACS.

In yet another embodiment of the present invention, the entire IFN-λ1cDNA was cloned into the pcDEF3 vector Goldman, et al., (1996)Biotechniques 21, 1013-1015, resulting in the plasmid pEF-IFN-λ1.Conditioned medium of COS-1 cells transiently transfected with thisexpression vector competed for binding with FL-IFN-λs and was positivein MHC class I induction experiments and in the EMSA experiments (FIG.21A), demonstrating that IFN-λs are secreted with their own signalpeptides.

In another embodiment of the present invention, recombinant IFN-λ1 wasproduced in E. coli. The recombinant IFN-λ1 produced in E. coli wasfound to be active in all the experiments described above (FIG. 21).

Example 9

In another embodiment of the present invention, the interaction betweenIFN-λ and its receptor was characterized by crosslinking (FIG. 22).Crosslinking experiments were performed with radiolabeled IFN-λ (FIGS.20B and C) and the IFN-λ receptor chains expressed in hamster cells(FIG. 22). COS cell-expressed FL-IFN-λ1-P was purified to homogeneityand radiolabeled (FIGS. 20B and C). The FL-IFN-λ1-P protein wastransiently expressed in COS cells and purified from conditioned mediaby immunoaffinity chromatography with the anti-FLAG M1 gel (Sigma)according to the manufacturer's suggested protocols. FL-IFN-λ1-P waslabeled with [32P]ATP and used for crosslinking to cells as described(Li, et al., (1989) Proc Natl Acad Sci USA 86, 558-562; Kotenko, et al.,(1995) J Biol Chem 270, 20915-20921; Pestka, et al., (1999) Protein ExprPurif 17, 203-214; Kotenko, et al., (2001) J Immunol 166, 7096-7103).

Parental hamster cells, cells expressing either the chimeric humanCRF2-12/γR1 chain, the intact human CRF24 chain, or both, were incubatedwith radiolabeled FL-IFN-λ1-P. The cells were washed to remove unboundligand, and bound ligand was crosslinked to the cells. Aftercrosslinking, the cells were lysed and crosslinked complexes wereresolved on 7.5% SDS-PAGE (FIG. 22).

The appearance of several labeled crosslinked complexes was observedonly in cells expressing both IFN-λ1 receptor chains. Neither parentalcells nor cells expressing either receptor chain alone were able to bindthe ligand. (FIG. 22, lanes 3, 4 and 5.)

Specificity of binding was shown by a competition assay with an excessof unlabeled IFN-λ1 (FIG. 22, lane 7). The major radiolabeled band inthe region of 35 kDa corresponds to free FL-IFN-λ1-P, and results fromligand bound to the cells but not crosslinked to receptors. A distinctcrosslinked complex migrating in the region of 200 kDa which is notpresent in the ligand crosslinked to itself in solution, likelyrepresents oligomers of IFN-λ1 and both receptor chains, formed as aresult of the association of the IFN-λ1 receptor chains induced byligand binding.

FL-IFN-λ1-P was also crosslinked to itself in solution to determinewhether it can oligomerize. Patterns of radiolabeled bands forcrosslinked or untreated ligand are identical (FIG. 22, lanes 1 and 2),strongly suggesting that IFN-λ1 is a monomer in solution. Thecrosslinking experiments demonstrated that IFN-λ1 requires the presenceof the both IFN-λ receptor chains on the cell surface and is unable tointeract with either chain expressed alone.

Thus, we identified a specific novel ligand-receptor complex, making afirst step in assigning functions for these novel cytokines. The CRF2-12protein can also be designated IFN-λR1 based on its function as the R1type subunit in the IFN-λ receptor complex.

Example 10 Signal Transduction and Biological Activity

Cytokines which utilize class II cytokine receptors for signalingprimarily activate the Jak-Stat signal transduction pathway (Kotenko, S.V. (2002) Cytokine Growth Factor Rev. 13, 223-240 and Kotenko, S. V. &Pestka, S. (2000) Oncogene 19, 2557-2565). Activation of this pathway isgenerally considered essential for induction of cytokine-responsivegenes.

To define which Stats are activated by the IFN-λs, we examined theactivation of several Stats in wild-type HT-29 cells and an HT-29 clone,1084, that overexpresses a chimeric receptor IL-10R1/IFN-λR1 (10R1/λR1)composed of the extracellular domain of the IL-10R1 chain linked to theintracellular domain of IFN-λR1 (CRF2-12). This chimeric receptor isable to bind IL-10 and dimerize with CRF24 (IL-10R2), a common secondchain for the IL-10, IL-22 and IFN-λ receptor complexes, to transduce asignal. The Stat proteins that are activated by signaling through thischimeric receptor upon IL-10 treatment represent those that areactivated by signaling through the intact IFN-λ receptor complex. Statactivation was measured by immunoprecipitating specific Stat proteinsfrom whole cell lysates, and then western blotting withphosphotyrosine-specific antibodies.

HT-29 cells do not normally express IL-10R1, therefore treatment withIL-10 did not induce activation of any Stats in these cells (FIGS. 23Aand B). However, these cells do express IL-22R1 (CRF2-9), and treatmentwith IL-22 resulted in the activation of Stat1, Stat2 (weakly), Stat3and Stat5. The parental HT-29 cell line Was also fully responsive toIFN-α because IFN-λ treatment activated Stat1, Stat2 and Stat3 in thesecells.

In 1084 cells, treatment with IL-10 induced activation (tyrosinephosphorylation) of Stat1, Stat2, Stat3 and Stat5.

Accordingly, since the chimeric receptor binds IL-10 and transduce anIFN-λ signal, in another embodiment of the present invention, modulationof the activity of the intracellular domain of CRF2-12, be it in achimeric receptor by binding of the ligand specific for theextracellular domain, or in a CRF2-12 receptor by binding by an IFN-λ,or another agonist or antagonist of the receptor, results in modulationof Stat1, Stat2, Stat3 and Stat5.

Activation of Stat2 is a unique feature of type I IFNs signaling. Thus,in another embodiment of the present invention, the pattern of Statactivation through the 10R1/λR1 chimeric receptor suggested thatsignaling through the IFN-λ receptor complex activates IFN-α-likeresponses.

Example 11

Type-I IFNs are unique insofar as they induce the activation of ISGF3.This transcription factor complex is composed of three subunits: Stat1,Stat2, and ISGF3γ (p48 or IRF-9, interferon regulatory factor 9).Activation of ISGF3 by type I IFNs induces expression of a distinctsubset of IFN-responsive genes (Darnell, et al., (1994) Science 264,1415-1421). To determine if IFN-λ activates ISGF3, we treated wild-typeHT-29 and A549 cells with IFN-λ and then measured ISGF3 activity usingan interferon-stimulated response element (ISRE) present in the proximalpromoter region of the ISG-15 gene. Treatment with IFN-λs, liketreatment with IFN-α, induces ISGF3 activity in HT-29 and A549 cells(FIGS. 23B and C). Accordingly, in accordance with another embodiment ofthe present invention, treatment with IFN-λs induces ISGF3 activity.

Example 12

IFN-λs also induce tyrosine phosphorylation of Stat1 and Stat2 in HT-29cells as measured by western blotting with anti-pY-Stat antibodies.Accordingly, in yet another embodiment of the present invention, IFN-λsinduce tyrosine phosphorylation of Stat1 and Stat2.

To determine whether IFN-λ and IFN-α possess overlapping biologicalactivities, HT29 colorectal adenocarcinoma, HeLa cervix adenocarcinoma,A549 lung carcinoma, and HaCaT keratinocyte cells were treated with bothligands and MHC class I antigen expression was evaluated. Both IFN-λ andIFN-α were able to up-regulate expression of MHC class I antigens tocomparable levels in all tested cell lines (FIG. 21A). Accordingly, inanother embodiment of the present invention, IFN-λ, agonist andantagonist thereof modulate expression of MHC class I antigens.

Example 13 IFN-λ1 Induces Cellular Antiviral Protection

Antiviral protection in response to IFN-λ1 was evaluated on HT29 cellsinfected with vesicular stomatitis virus (FIG. 24A, row C). IFN-α wasused as a positive control (FIG. 24A, row A). Equal amount of cells wereplated in all wells and treated with two-fold serial dilutions ofindicated ligands (FIG. 24A, rows A and C). Twenty-four hours later thevirus was added in all wells except for the first seven wells in row F.As can be seen from FIG. 24A, IFN-λ1 was more effective than IFN-α inprotecting cells from viral infection.

Accordingly, in another embodiment of the present invention, IFN-λs,alone or in combination, other agonists of CRF2-12, and modulators ofthe intracellular domain of CRF2-12, are effective in providingprotection from viral infection. In another embodiment of the presentinvention, IFN-λ1, IFN-λ2 or IFN-λ3, or effective combinations thereof,induces overlapping signaling and biological activities with those ofIFN-α□ in intact cells expressing an effective receptor.

Example 14 Expression of IFN-λs mRNA was Evaluated by RT-PCR inVirus-Infected Cells

HeLa cells were left untreated (lane 3) or infected with either Denguevirus (DV, lanes 4-6) Sindbis virus (SV, lanes 7-9) or vesicularstomatitis virus (VSV, lanes 10-12). HT29 cells were left untreated(lane 13) or infected with VSV (lanes 14-16). HuH7 cells were leftuntreated (lane 17) or infected with DV (lanes 18-20) or SV (lanes21-23). Cells were collected at post-infection times shown in theFigure, RNA was isolated and RT-PCR was performed as described inMaterials and Methods. Water was used as a negative control for RT-PCR(lane 2). 1 kb ladder was run in lanes 1 and 24.

The expression of IFN-λ1, IFN-λ2 and IFN-λ3 mRNAs was detected in anumber of cell lines infected with various viruses (FIG. 24B).

It was also found that dendritic cells, which are important producers ofIFN-α (Siegal, et al., (1999) Science 284, 1835-1837) expressed IFN-λ1mRNA in response to treatment with polyI:C.

Accordingly, in another embodiment of the present invention, thepresence and/or quantity of IFN-λ1, IFN-λ2 and/or IFN-λ3 present, ormRNA expressed, is an indicator of infection, in particular viralinfection. In another aspect of the present invention, IFN-λ expressionis modulated in response to viral infections. In another embodiment ofthe present invention, the cytokines designated IFN-λs play a functionalIFN-α independent role in an antiviral cellular defense.

Example 15 Signaling Through the CRF2-12 Intracellular Domain InducesApoptosis

We also evaluated whether biological activities induced by IFN-λsthrough the intact IFN-λ receptor complex composed of CRF2-12 and CRF2-4chains, can be reproduced in cells expressing the FL-IL-10R1/CRF2-12chimeric receptor upon IL-10 treatment. HT-29 cells expressingFL-IL-10R1/CRF2-12 were treated with various amounts of IL-10. Threedays later the cells were collected to measure the level of MHC class Iantigen expression. Surprisingly, cells treated with IL-10 atconcentration of about 10 ng/ml and higher, appeared round-shaped, notattached to the plastic, and apparently did not proliferate, suggestingthat the cells underwent apoptosis in response to IL-10. None of thesechanges were observed in untreated cells, in cells treated with IL-22 orIFN-γ, or in parental HT-29 cells treated with IL-10.

The induction of apoptosis in HT-29 cells expressing FL-IL-10R1/CRF2-12chain by IL-10 was subsequent confirmed: i) by staining apoptotic cellswith propidium iodide, and ii) by demonstrating phosphatidylserineexternalization in apoptotic cells with the use of Anexin V-FITC andflow cytometry. The induction of apoptosis in these cells by IL-10 wasdependent on the concentration of IL-10 and on the level of theFL-IL-10R1/CRF2-12 chain expression. Apoptotic effects were stronger athigher concentrations of IL-10 and in cell clones with higher level ofFL-IL-10R1/CRF2-12 expression.

At lower concentration of IL-10 (about few ng/ml) cells demonstratedantiproliferative response and at concentration of IL-10 about 1 ng/mldemonstrated activities characteristic for IFN-λ (MHC class I antigeninduction and antiviral activity).

Thus, with declining concentration of IL-10, various activities can beinduced in cells starting from apoptosis, followed by antiproliferativeeffect and the induction of antiviral state. Accordingly, anotherembodiment of the present invention results that either apoptosis,antiproliferative effect or antiviral protection may be obtained byeffectively modulating the activity of the intracellular domain ofCRF2-12.

In all cells tested so far, the level of CRF2-12 expression appeared tobe low. Thus, the fact that in all tested cell lines IFN-λ appeared toinduce can be explained by the low level of CRF2-12 expression.Accordingly, in another embodiment of the present invention, whenviruses or other natural or pharmaceutical agents modulate the level ofCRF2-12 expression, IFN-λ, or other compositions that modulateeffectively the activity of the intracellular domain of CRF2-12, may beused to induce antiviral protection, apoptosis or to have anantiproliferative effect.

Example 16 Cloning of CRF2-11 cDNA

CRF2-11 cDNA was obtained by PCR as follows.

First Round Primers:

5′-TTTGGAAAGAAACAATGTTCTAGGTC-3′ (R11-2F) and 5′-CTTCACCTGGGCCCTTCCGC-3′(R11-7R)

were used to amplify a libraries containing cDNA isolated from humanleukocytes (Clontech, catalog #HL4050AH)

Second Round Primers:

5′-CTGAGGGTACCAAATGCAGACTTTCACAATG-3′ (R11-3F) and5′-GGGAATTCATGAGATCCAGGCCCTGAGGAGTTC-3′ (R11-6R)

and the PCR product of the first round as a template.

Example 17

In another embodiment of the present invention IFN-λ1 induces cellularantiviral protection. As shown in FIG. 26A, IFN-λ1 induced antiviralprotection in HT29, A549 and HaCaT cells infected with vesicularstomatitis virus (VSV, an RNA rhabdovirus) and in HT29 cells infectedwith encephalomyocarditis virus (EMCV, an RNA picornavirus). Inantiviral assays IFN-λ1 demonstrated antiviral potency comparable withthat of IFN-α, on the order of 10⁷-10⁸ IFN-α-relevant units per mg.Moreover, when HT29 cells were pretreated with 500 pg/ml of IFN-λ1 priorto VSV infection, the titer of VSV in the conditioned media of infectedcells (48 h after infection) dropped more then 3 orders of magnitude incomparison to the VSV titer produced by control untreated cells. Incells pretreated with 1250 pg/ml the viral titer dropped more then 6orders of magnitude as determined by viral killing curve (data notshown). Accordingly, in another aspect of the present invention IFN-λsare used to induce antiviral protection.

In accordance with another embodiment of the present invention, IFN-λ1is capable of inducing transcription of type I IFN-responsiveISRE-controlled genes encoding 2′,5′-OAS and MxA protein (FIG. 26B).Thus, in intact cells, IFN-λ induces overlapping signaling andbiological activities with those of IFN-α. These signaling andbiological activities, include, for example, upregulation of MHC class Iantigen expression, induction of antiviral protection, and induction ofIFN-regulated genes. Accordingly, in another aspect of the presentinvention IFN-λs are used to induce expression of a protein thatmediates antiviral protection. These proteins may include, for example,2′,5′-oligoadenylate synthetase (2=,5=-OAS) and Mx proteins.

In another embodiment of the present invention HT29 cells or A549 cellsinfected with EMCV secrete IFN-λ specific activity capable ofstimulating STAT1 activation through the CRF2-12-λR1 and CRF2-4heterodimer (FIG. 26C). Uninfected cells, in accordance with the presentinvention, do not produce IFN-λ activity. Accordingly, in another aspectof the present invention IFN-λs are used to detect the presence ofinfected cells. In yet another embodiment of the present invention,antagonists are used to modulate inflammation.

Other Materials and Methods

See, for example, Kotenko, et al., Nature Immunology 4:69-77 (2003),which is incorporated by reference herein in its entirety.

Cells, Transfection and Cytofluorographic Analysis.

The 16-9 hamster x human somatic cell hybrid line is the Chinese hamsterovary cell (CHO-K1) hybrid containing a translocation of the long arm ofhuman Chromosome 6 encoding the human IFNGR1 (Hu-IFN-γR1) gene and atransfected human HLA-B7 gene (Soh, J., Donnelly, R. J., Mariano, T. M.,Cook, J. R., Schwartz, B. & Pestka, S. (1993) Proc. Natl. Acad. Sci.U.S.A. 90, 8737-8741). The cells were maintained in F12 (Ham) medium(Sigma) containing 10% heat-inactivated fetal bovine serum (Sigma).COS-1 cells, an SV40 transformed fibroblast-like simian CV-1 cell line,were maintained in DMEM medium (GibcoBRL) with 10% heat-inactivatedfetal bovine serum.

Colorectal adenocarcinoma HT29 cells were maintained in DMEM medium(GibcoBRL) with 10% heat-inactivated fetal bovine serum.

Cells were transfected as previously described (Kotenko, et al., (1997)EMBO J. 16, 5894-5903 and Kotenko, et al., (2000) Proc Natl Acad Sci USA97, 1695-1700). COS cell supernatants were collected at 72 hrs as asource of the expressed proteins.

To detect cytokine-induced MHC class I antigen (HLA-B7) expression,cells were treated with COS cell supernatants or purified recombinantproteins as indicated in the text for 72 hours and analyzed by flowcytometry.

Cell surface expression of the HLA-B7 antigen was detected by treatmentwith mouse anti-HLA (W6/32) (Barnstable, et al., (1978) Cell 14,9-20)-monoclonal antibody followed by fluoresceinisothiocyanate-conjugated goat anti-mouse IgG (Santa Cruz BiotechnologyInc.). The cells then were analyzed by cytofluorography as previouslydescribed (Kotenko, et al., (1999) Proc Natl Acad Sci USA 96,5007-5012).

Unless otherwise specified, whether a result is statisticallysignificant, means that when appropriately measured in any effectivemanner, there is a probability of less than 50% that the result occurredby mere chance. Other preferred confidence levels include 70%, 75%, 80%,85%, 90%, 95% and 99%.

Electrophoretic mobility shift assays (EMSAs) and Western and Northernblotting.

Cells were starved overnight in serum-free media and then treated withIL-10 or IL-22 as indicated in the text for 15 minutes at 37° C. andused for EMSA experiments to detect activation of Stat1, Stat3 and Stat5as described (Kotenko, S. V., Krause, C. D., Izotova, L. S., Pollack, B.P., Wu, W. & Pestka, S. (1997) EMBO J. 16, 5894-5903). EMSAs wereperformed with a 22-base pair sequence containing a Stat1α binding sitecorresponding to the GAS element in the promoter region of the humanIRF-1 gene (5′-GATCGATTTCCCCGAAATCATG-3) as described (Kotenko, et al.(1995) J Biol Chem 270, 20915-20921 and Kotenko, et al., (1996) J BiolChem 271, 17174-17182).

1. An isolated nucleic acid molecule comprising a polynucleotide havinga nucleotide sequence selected from the group consisting of: (a) anucleotide sequence encoding the CRF2-12 polypeptide having the completeamino acid sequence in SEQ ID NO: 2; (b) a nucleotide sequence encodingresidues 2-520 of SEQ ID NO: 2; (c) a nucleotide sequence encodingresidues 19-520 in SEQ ID NO: 2; (d) a nucleotide sequence encoding theCRF2-12 polypeptide extracellular domain; (e) a nucleotide sequenceencoding the extracellular domain of the CRF2-12 protein in plasmidpEF-CRF2-12/γR1; (f) a nucleotide sequence encoding the extracellulardomain of the CRF2-12 protein in plasmid pEF3-FLγR2/αR2c; (g) anucleotide sequence encoding the CRF2-12 polypeptide intracellulardomain; (h) a nucleotide sequence encoding the intracellular domain ofthe CRF2-12 protein in plasmid pEF-CRF2-12; (i) a nucleotide sequenceencoding the intracellular domain of the CRF2-12 protein in plasmidpEF-FLCRF2-12; and (j) a nucleotide sequence complementary to any ofnucleotide sequences (a)-(i).
 2. An isolated nucleic acid moleculecomprising a polynucleotide having a nucleotide sequence selected fromthe group consisting of: (a) a nucleotide sequence encoding a chimericreceptor comprising the extracellular domain of CRF2-12 and theintracellular domain of another membrane bound receptor; (b) anucleotide sequence encoding a chimeric receptor comprising theextracellular domain of CRF2-12 and the intracellular domain of anothermembrane bound tyrosine kinase receptor; (c) a nucleotide sequenceencoding a chimeric receptor comprising the extracellular domain ofCRF2-12 and the intracellular domain of a cytokine receptor; (d) anucleotide sequence encoding a chimeric receptor comprising theextracellular domain of CRF2-12 and the intracellular domain of an IFNR1 type receptor; (e) a nucleotide sequence encoding a chimeric receptorcomprising the extracellular domain of CRF2-12 and the intracellulardomain of IFNγR1 ; (f) a nucleotide sequence encoding the chimericprotein in plasmid pEF-CRF2-12/γR1; and (g) a nucleotide sequencecomplementary to any of nucleotide sequences (a)-(f).
 3. An isolatednucleic acid molecule comprising a polynucleotide having a nucleotidesequence selected from the group consisting of: (a) a nucleotidesequence encoding a chimeric receptor comprising the intracellulardomain of CRF2-12 and the extracellular domain of another membrane boundreceptor; (b) a nucleotide sequence encoding a chimeric receptorcomprising the intracellular domain of CRF2-12 and the extracellulardomain of another membrane bound tyrosine kinase receptor; (c) anucleotide sequence encoding a chimeric receptor comprising theintracellular domain of CRF2-12 and the extracellular domain of acytokine receptor; (d) a nucleotide sequence encoding a chimericreceptor comprising the intracellular domain of CRF2-12 and theextracellular domain of an IFN R1 type receptor; (e) a nucleotidesequence encoding a chimeric receptor comprising the intracellulardomain of CRF2-12 and the extracellular domain of IL-10R1; (f) anucleotide sequence encoding the chimeric protein in plasmidpEF-IL-10R1/IFN-λR1; and (g) a nucleotide sequence complementary to anyof nucleotide sequences (a)-(f).
 4. An isolated nucleic acid moleculecomprising a tandem vector comprising a nucleotide sequence selectedfrom the group consisting of: (a) a nucleotide sequence encoding tworeceptors, wherein the first receptor is an R1 type receptor and thesecond receptor is an R2 type receptor, and wherein the expression ofeach receptor is controlled by separate promoters and polyadenylationsignals; (b) a nucleotide sequence encoding two receptors, wherein thefirst receptor comprises the extracellular domain of CRF2-12 and thesecond receptor is an R2 type receptor, and wherein the expression ofeach receptor is controlled by separate promoters and polyadenylationsignals; (c) a nucleotide sequence encoding two receptors, wherein thefirst receptor comprises the intracellular domain of CRF2-12 and thesecond receptor is an R2 type receptor, and wherein the expression ofeach receptor is controlled by separate promoters and polyadenylationsignals; (d) a nucleotide sequence encoding two receptors, wherein thefirst receptor comprises CRF2-12 and the second receptor is an R2 typereceptor, and wherein the expression of each receptor is controlled byseparate promoters and polyadenylation signals; (e) any of tandemvectors (a)-(d), wherein the R2 type receptor comprises CRF2-4; (f) thetandem vector pEF-CRF2-12/γR1+CRF2-4; and (g) a nucleotide sequencecomplementary to any of nucleotide sequences (a)-(f).
 5. (canceled) 6.An isolated nucleic acid molecule comprising a polynucleotide encodingat least a portion of an IFN-λ2 polypeptide having the complete aminoacid sequence shown in SEQ ID NO
 10. 7. An isolated nucleic acidmolecule comprising a polynucleotide encoding at least a portion of anIFN-λ3 polypeptide having the complete amino acid sequence shown in SEQID NO
 12. 8. (canceled)
 9. An isolated nucleic acid molecule comprisinga polynucleotide comprising a nucleotide sequence selected from thegroup consisting of: (a) a nucleotide sequence encoding the IFN-λ2polypeptide having the complete amino acid sequence in SEQ ID NO: 10;(b) a nucleotide sequence encoding residues 2-196 of SEQ ID NO: 10; (c)a nucleotide sequence encoding residues 23-196 of SEQ ID NO: 10; (d) anucleotide sequence encoding residues 12-196 of SEQ ID NO: 10; (e) anucleotide sequence encoding residues 13-196 of SEQ ID NO: 10; (f) anucleotide sequence encoding residues 31-196 of SEQ ID NO: 10; (g) anucleotide sequence encoding residues 92-196 of SEQ ID NO: 10; (h) anucleotide sequence encoding residues 93-196 of SEQ ID NO: 10; (i) anucleotide sequence encoding residues 113-196 of SEQ ID NO: 10; (j) thenucleotide sequence of the genomic fragment encoding the complete IFN-λ2gene contained in plasmid pEF-FL-IFN-λ2 gene; (k) a nucleotide sequenceencoding a polypeptide encoded by the cDNA contained in plasmidpEF-FL-IFN-λ2; (1) a nucleotide sequence encoding the mature polypeptideencoded by the cDNA contained in clone pEF-FL-IFN-λ2; and (m) anucleotide sequence complementary to any of nucleotide sequences(a)-(l).
 10. An isolated nucleic acid molecule comprising apolynucleotide comprising a nucleotide sequence selected from the groupconsisting of: (a) a nucleotide sequence encoding the IFN-λ3 polypeptidehaving the complete amino acid sequence in SEQ ID NO: 12; (b) anucleotide sequence encoding residues 2-196 of SEQ ID NO: 12; (c) anucleotide sequence encoding residues 23-196 of SEQ ID NO: 12; (d) anucleotide sequence encoding residues 6-196 of SEQ ID NO: 12; (e) anucleotide sequence encoding residues 12-196 of SEQ ID NO: 12; (f) anucleotide sequence encoding residues 7-196 of SEQ ID NO: 12; (g) anucleotide sequence encoding residues 13-196 of SEQ ID NO: 12; (h) anucleotide sequence encoding residues 31-196 of SEQ ID NO: 12; (i) thenucleotide sequence of the genomic fragment encoding the complete IFN-λ3gene contained in plasmid pEF-FL-IFN-λ3gene; (j) a nucleotide sequenceencoding a polypeptide encoded by the cDNA contained in plasmidpEF-FL-IFN-λ3; (k) a nucleotide sequence encoding a mature polypeptideencoded by the cDNA contained in plasmid pEF-FL-IFN-λ3; and (l) anucleotide sequence complementary to any of nucleotide sequences(a)-(k).
 11. A host cell containing a recombinant vector comprising anucleic acid having a sequence selected from the sequences in claims1-4, 6-7 and 9-10.
 12. (canceled)
 13. An isolated polypeptide comprisingan amino acid sequence selected from the group consisting of: thecomplete amino acid sequence of SEQ ID NO: 10; amino acids 2-196 of SEQID NO: 10; amino acids 23-196 of SEQ ID NO: 10; amino acids 12-196 ofSEQ ID NO: 10; amino acids 13-196 of SEQ ID NO: 10; amino acids 31-196of SEQ ID NO: 10; amino acids 92-196 of SEQ ID NO: 10; amino acids93-196 of SEQ ID NO: 10; amino acids 113-196 of SEQ ID NO: 10; a IFN-λ2polypeptide encoded by plasmid pEF-FL-IFN-λ2 gene; a polypeptide encodedby the cDNA contained in plasmid pEF-FL-IFN-λ2; and a mature polypeptideencoded by the cDNA contained in clone pEF-FL-IFN-λ2.
 14. (canceled) 15.An isolated antibody that binds specifically to an epitope comprising apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 10 and SEQ ID NO:
 12. 16. Acomposition comprising an IFN-λ-2 polypeptide selected from and apharmaceutically acceptable carrier.
 17. A composition comprising acombination of IFN-λ1, IFN-λ2 and IFN-λ3 polypeptides, wherein the ratioof IFN-λ1:IFN-λ2:IFN-λ3 is described by the formula m:n:o, wherein m maybe any number between 0 and 1000, n may be any number between 0 and 1000and 0 may be any number between 0 and 1000, and wherein at least one ofm, n or o is different from
 0. 18. A composition comprising an IFN-λ2antibody or an IFN-λ3 antibody and a pharmaceutically acceptablecarrier.
 19. A composition comprising a combination of anti-IFN-λ1specific antibody, anti-IFN-λ2 specific antibody and anti-anti-IFN-λ3specific antibody, wherein the ratio of anti-IFN-λ1 specific antibody:anti-TN-λ2 specific antibody: anti-IFN-λ3 specific antibody is describedby the formula x:y:z, wherein x may be any number between 0 and 1000, ymay be any number between 0 and 1000 and z may be any number between 0and 1000, and wherein at least one of x, y or z is different from
 0. 20.A composition comprising an antibody specific for the extracellulardomain of IFN-λR1.
 21. (canceled)
 22. A method for identifying compoundscapable of enhancing or inhibiting a biological activity of IFN-λ2comprising i) contacting a receptor which is activated by IFN-λ2 withthe candidate compound in the presence and in the absence of IFN-λ2; ii)assaying for receptor activity in the presence of the candidate compoundand IFN-λ2; iii) assaying for receptor activity in the presence of thecandidate compound and the absence of IFN-λ2; and iv) comparing theactivity to a standard level of activity, the standard being assayedwhen contact is made between the receptor and IFN-λ2 in the absence ofthe candidate compound.
 23. A method for identifying compounds capableof enhancing or inhibiting a biological activity of IFN-λ3 comprising i)contacting a receptor which is activated by IFN-λ3 with the candidatecompound in the presence and in the absence of IFN-λ3; ii) assaying forreceptor activity in the presence of the candidate compound and IFN-λ3;iii) assaying for receptor activity in the presence of the candidatecompound and the absence of IFN-λ3; and iv) comparing the activity to astandard level of activity, the standard being assayed when contact ismade between the receptor and IFN-λ3 in the absence of the candidatecompound.
 24. A method for identifying compounds that block, on areagonists or antagonists of CRF2-12 comprising i) contacting a receptorcomprising CRF2-12 with a candidate compound in the presence and in theabsence of an IFN-λ composition; ii) assaying for receptor activity inthe presence of the candidate compound and the IFN-λ composition; iii)assaying for receptor activity in the presence of the candidate compoundand the absence of the IFN-λ composition; and iv) comparing the activityto a standard level of activity, the standard being assayed when contactis made between the receptor and the IFN-λ composition in the absence ofthe candidate compound.
 25. The method of claim 23 wherein the IFN-λcomposition comprises IFN-λ2 or IFN-λ3 and a pharmaceutically acceptablecarrier.
 26. A diagnostic method useful during diagnosis of a disordercomprising i) assaying the expression level of an mRNA or polypeptideselected from IFN-λ2, IFN-λ3 and IFN-λR1 in cells or body fluid of anindividual; and ii) comparing the expression level with a standardexpression level, whereby an increase or decrease in the assayedexpression level compared to the standard expression level is indicativeof a disorder
 27. A method for treating an individual in need of anincreased level of interferon activity in the body comprisingadministering to such an individual a composition comprising atherapeutically effective amount of a composition of claim 16, 17 or 18.28. A method for treating an individual in need of a decreased level ofinterferon activity in the body comprising administering to such anindividual a composition comprising a therapeutically effective amountof a composition of claim
 17. 29. A method of treating an individual inneed of an increased level of activation of a receptor complexcomprising CRF2-12, wherein the method comprises administering to suchan individual a composition comprising a therapeutically effectiveamount of an agonist of the receptor complex comprising CRF2-12.
 30. Amethod of treating an individual in need of a decreased level ofactivation of a receptor complex comprising CRF2-12, wherein the methodcomprises administering to such an individual a composition comprising atherapeutically effective amount of an antagonist of the receptorcomplex comprising CRF2-12.
 31. A method for testing a candidatecomposition as an antagonist or agonist of the IFN-λR1 receptor complex,wherein the method comprises: i) assaying for receptor activity in thepresence of a negative control composition; ii) assaying for receptoractivity in the presence of a positive control composition; iii)assaying for receptor activity in the presence of the candidatecomposition; iv) assaying for receptor activity in the presence of thecandidate composition and the positive control composition; wherein theresulting activity from ii) is statistically greater than the resultingactivity from i); wherein the candidate composition is considered anagonist if the resulting activity from iii) is statistically greaterthan the resulting activity from i); wherein the candidate compositionis considered an antagonist if the resulting activity from iv) isstatistically smaller than the resulting activity from ii); wherein thecandidate composition is considered neither an agonist nor an antagonistif the resulting activity from iv) is not statistically different fromthe resulting activity from ii) and the resulting activity from iii) isnot statistically different from the resulting activity from i).
 32. Themethod of claim 31 wherein the positive control composition comprises acomposition selected from the group consisting of IFN-λ2, IFN-λ3 and thecomposition of claim
 17. 33. (canceled)
 34. The method of claim 32wherein the IFN-λ2 composition is selected from the group consisting of:the complete amino acid sequence of SEQ ID NO: 10; amino acids 2-196 ofSEQ ID NO: 10; amino acids 23-196 of SEQ ID NO: 10; amino acids 12-196of SEQ ID NO: 10; amino acids 13-196 of SEQ ID NO: 10; amino acids31-196 of SEQ ID NO: 10; amino acids 92-196 of SEQ ID NO: 10; aminoacids 93-196 of SEQ ID NO: 10; amino acids 113-196 of SEQ ID NO: 10; aIFN-λ2 polypeptide encoded by plasmid pEF-FL-IFN-λ2gene; a polypeptideencoded by the cDNA contained in plasmid pEF-FL-IFN-λ2; and a maturepolypeptide encoded by the cDNA contained in clone pEF-FL-IFN-λ2. 35.The method of claim 32 wherein the IFN-λ3 composition is selected fromthe group consisting of: a polypeptide having the complete amino acidsequence of SEQ ID NO: 12; amino acids 2-196 of SEQ ID NO: 12; aminoacids 23-196 of SEQ ID NO: 12; amino acids 6-196 of SEQ ID NO: 12; aminoacids 12-196 of SEQ ID NO: 12; amino acids 7-196 of SEQ ID NO: 12; aminoacids 13-196 of SEQ ID NO: 12; amino acids 31-196 of SEQ ID NO: 12; anIFN-λ3 polypeptide encoded by plasmid pEF-FL-IFN-λ3gene; a polypeptideencoded by the cDNA contained in plasmid pEF-FL-IFN-λ3; and a maturepolypeptide encoded by the cDNA contained in clone pEF-FL-IFN-λ3.